Anti-hemagglutinin antibodies and methods of use

ABSTRACT

The present invention provides anti-hemagglutinin antibodies, compositions comprising anti-hemagglutinin antibodies, and methods of using the same.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/725,859, filed on 13 Nov. 2012, which is incorporated by referenceherein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 22, 2013, isnamed P4982R1_US_SL.txt and is 222,627 bytes in size.

FIELD OF THE INVENTION

The present invention provides anti-hemagglutinin antibodies,compositions comprising anti-hemagglutinin antibodies, and methods ofusing the same.

BACKGROUND

Influenza virus infection causes between three and five million cases ofsevere illness and between 250,000 and 500,000 deaths every year aroundthe world. In the United States alone, 5% to 20% of the populationbecomes infected with influenza virus each year, with the majority ofthese infections caused by the influenza A virus. (See, e.g., Dushoff etal., (2006) Am J Epidemiology 163:181-187; Thompson et al., (2004) JAMA292:1333-1340; Thompson et al., (2003) JAMA 289:179-186.) Approximately200,000 people in the United States become hospitalized withinfluenza-related complications every year, resulting in 7,000 to 30,000deaths annually. The burden associated with influenza virus infection onhealth care costs and lost productivity is extensive. Hospitalizationand deaths mainly occur in high-risk groups, such as the elderly,children, and chronically ill.

Influenza viruses are segmented membrane-enveloped negative-strand RNAviruses belonging to the Orthomyxoviridae family. Influenza A virusconsists of 9 structural proteins and 1 non-structural protein, whichinclude three virus surface proteins: hemagglutinin (HA or H),neuraminidase (NA or N), and matrix protein 2 (M2). The segmented natureof the influenza viral genome allows the mechanism of geneticreassortment (i.e., exchange of genome segments) to take place duringmixed infection of a cell with different influenza viral strains Annualepidemics of influenza occur when the antigenic properties of the viralsurface proteins hemagglutinin and neuraminidase are altered. Themechanism of altered antigenicity is twofold: antigenic shift, caused bygenetic rearrangement between human and animal viruses afterco-infection of host cells with at least two viral subtypes, which cancause a pandemic; and antigenic drift, caused by small changes in thehemagglutinin and neuraminidase proteins on the virus surface, which cancause influenza epidemics.

Influenza A viruses may be further classified into various subtypesdepending on the different hemagglutinin and neuraminidase viralproteins displayed on their surface. Each influenza A virus subtype isidentified by the combination of its hemagglutinin and neuraminidaseproteins. There are 16 known HA subtypes (H1-H16) and 9 known NAsubtypes (N1-N9). The 16 hemagglutinin subtypes are further classifiedinto two phylogenetic groups: Group1 includes hemagglutinin H1, H2, H5,H6, H8, H9, H11, H12, H13, and H16 subtypes; Group2 includeshemagglutinin H3, H4, H7, H10, H14, and H15 subtypes.

Hemagglutinin promotes viral attachment and entry into the host cell;neuraminidase is required for viral budding from the infected cell. Thehemagglutinin of influenza A virus comprises two structurally distinctregions—a globular head region and a stalk or stem region. The globularhead region contains a receptor binding site which is responsible forvirus attachment to a target cell. The stalk (or stem) region ofhemagglutinin contains a fusion peptide which is necessary for membranefusion between the viral envelope and an endosomal membrane of theinfected cell. (See, e.g., Bouvier and Palese (2008) Vaccine 26 Suppl 4:D49-53; Wiley et al., (1987) Ann Rev Biochem 556:365-394.)

Current treatment for influenza virus infection includes neuraminidaseinhibitors, such as oseltamivir and zanamivir. Oseltamivir is a widelyused prophylactic and early therapeutic treatment option for influenza Avirus infection. (See, e.g., Kandel and Hartshorn (2001) BioDrugs:Clinical Immunotherapy, Biopharmaceuticals and Gene Therapy 15:303-323;Nicholson et al., (2000) Lancet 355:1845-1850; Treanor et al., (2000)JAMA 283:1016-1024; and Welliver et al., (2001) JAMA 285:748-754.)However, oseltamivir treatment must begin within 48 hours of symptomonset to provide a significant clinical benefit. (See, e.g., Aoki et al(2003) J Antimicrobial Chemotherapy 51:123-129.) This liabilitycompromises oseltamivir's ability to treat severely ill patients, whoare typically beyond the optimal 48-hour treatment window at the time ofseeking treatment. Therefore, significant focus has recently been placedon identifying influenza virus therapeutics to treat hospitalizedinfluenza virus infected patients. One strategy has focused ondevelopment of human monoclonal antibodies (mAbs) that target a highlyconserved epitope on the stalk of influenza A virus hemagglutinin. (See,e.g., Corti et al., (2011) Science 333:850-856; Ekiert et al., (2009)Science 324:246-251; Ekiert et al., (2011) Science 333:843-850; Sui etal., (2009) Nature Structural & Molecular Biology 16:265-273; Dreyfus etal., (2012) Science 337:1343-1348; Wu et al., (2012) J Virology2012.09.034; Clementi et al., (2011) PLoS One 6:1-10. See alsoInternational Patent Application Publication Nos: WO2009/115972,WO2011/117848, WO2008/110937, WO2010/010466, WO2008/028946,WO2010/130636, WO2012/021786, WO2010/073647, WO2011/160083,WO2011/111966, WO2002/46235, and WO2009/053604; U.S. Pat. Nos. 5,631,350and 5,589,174.)

Several reports have described monoclonal antibodies (mAb) that bindhemagglutinin and broadly neutralize influenza A virus. For example,Corti et al. (supra) described antibody FI6v3, which was cloned from ahuman plasma cell and shown to neutralize human influenza A virusesbelonging to both Group1 and Group2 hemagglutinin subtypes. The FI6v3mAb was discovered as a result of a heroic effort of analyzingapproximately 104,000 human plasma cells. Additionally, Dreyfus et al.(supra) recently described the identification of antibody CR9114 byphage display panning; antibody CR9114 was shown to bind to a highlyconserved stalk epitope shared between influenza A virus and influenza Bvirus hemagglutinin.

Despite these reports, a need still exists in the art for novelinfluenza A virus therapies effective against Group1 and Group2influenza A virus subtypes. The present invention meets this need andprovides other benefits for the treatment of influenza A virusinfection.

SUMMARY OF THE INVENTION

The present invention provides anti-hemagglutinin antibodies,compositions comprising anti-hemagglutinin antibodies, and methods ofusing the same.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises three heavy chain hypervariable regions(HVR-H1, HVR-H2, and HVR-H3) and three light chain hypervariable regions(HVR-L1, HVR-L2, and HVR-L3), wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:178;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:179;    -   (c) HVR-H3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:180 and 181;    -   (d) HVR-L1 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:182, 183, 184, 185, and 186;    -   (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO:187;        and    -   (f) HVR-L3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:188, 189, and 190.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, three, four,five and/or six hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:178;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:179;    -   (c) HVR-H3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:180 and 181;    -   (d) HVR-L1 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:182, 183, 184, 185, and 186;    -   (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO:187;        and    -   (f) HVR-L3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:188, 189, and 190.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising three light chain hypervariableregions (HVR-L1, HVR-L2, and LVR-L3), wherein:

-   -   (a) HVR-L1 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:182, 183, 184, 185, and 186;    -   (b) HVR-L2 comprises the amino acid sequence of SEQ ID NO:187;        and    -   (c) HVR-L3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:188, 189, and 190.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising three heavy chain hypervariableregions (HVR-H1, HVR-H2, and HVR-H3), wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:178;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:179;        and    -   (c) HVR-H3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:180 and 181.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, and/or threelight chain hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-L1 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:182, 183, 184, 185, and 186;    -   (b) HVR-L2 comprises the amino acid sequence of SEQ ID NO:187;        and    -   (c) HVR-L3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:188, 189, and 190.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, and/or threeheavy chain hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:178;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:179;        and    -   (c) HVR-H3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:180 and 181.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain variable region and a lightchain variable region, wherein the heavy chain variable region comprisesan amino acid sequence selected from the group consisting of SEQ IDNOs:111 and 115, and the light chain variable region comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs:113, 117,119, 122, 124, 126, 128, 130, and 132.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a light chain variable region comprising anamino acid sequence selected from the group consisting of SEQ IDNOs:113, 117, 119, 122, 124, 126, 128, 130, and 132.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain variable region comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:111and 115.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain and a light chain, wherein theheavy chain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs:110, 114, and 120, and the light chaincomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs:112, 116, 118, 121, 123, 125, 127, 129, and 131.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a light chain comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:112, 116, 118,121, 123, 125, 127, 129, and 131.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:110, 114, and120.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises three heavy chain hypervariable regions(HVR-H1, HVR-H2, and HVR-H3) and three light chain hypervariable regions(HVR-L1, HVR-L2, and HVR-L3), wherein:

-   -   (a) HVR-H1 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:191 and 192;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:193;    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:194;    -   (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO:195;    -   (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO:196;        and    -   (f) HVR-L3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:197, 198, and 199.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, three, four,five and/or six hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-H1 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:191 and 192;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:193;    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:194;    -   (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO:195;    -   (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO:196;        and    -   (f) HVR-L3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:197, 198, and 199.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising three light chain hypervariableregions (HVR-L1, HVR-L2, and LVR-L3), wherein:

-   -   (a) HVR-L1 comprises the amino acid sequence of SEQ ID NO:195;    -   (b) HVR-L2 comprises the amino acid sequence of SEQ ID NO:196;        and    -   (c) HVR-L3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:197, 198, and 199.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising three heavy chain hypervariableregions (HVR-H1, HVR-H2, and HVR-H3), wherein:

-   -   (a) HVR-H1 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:191 and 192;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:193;        and    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:194.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, and/or threelight chain hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-L1 comprises the amino acid sequence of SEQ ID NO:195;    -   (b) HVR-L2 comprises the amino acid sequence of SEQ ID NO:196;        and    -   (c) HVR-L3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:197, 198, and 199.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, and/or threeheavy chain hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-H1 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs:191 and 192;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:193;        and    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:194.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain variable region and a lightchain variable region, wherein the heavy chain variable region comprisesan amino acid sequence selected from the group consisting of SEQ IDNOs:134, 138, 142, 148, and 234, and the light chain variable regioncomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs:136, 140, 144, 146, 150, 152, and 235.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a light chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:136, 140, 144, 146, 150, 152, and 235.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain variable region comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:134, 138, 142, 148, and 234.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain and a light chain, wherein theheavy chain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs:133, 137, 141, and 147, and the light chaincomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs:135, 139, 143, 145, 149, and 151.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a light chain comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 135, 139,143, 145, 149, and 151.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 133, 137,141, and 147.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises three heavy chain hypervariable regions(HVR-H1, HVR-H2, and HVR-H3) and three light chain hypervariable regions(HVR-L1, HVR-L2, and HVR-L3), wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:200;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:201;    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:202;    -   (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO:203;    -   (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO:204;        and    -   (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO:205.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, three, four,five and/or six hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:200;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:201;    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:202;    -   (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO:203;    -   (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO:204;        and    -   (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO:205.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising three light chain hypervariableregions (HVR-L1, HVR-L2, and LVR-L3), wherein:

-   -   (a) HVR-L1 comprises the amino acid sequence of SEQ ID NO:203;    -   (b) HVR-L2 comprises the amino acid sequence of SEQ ID NO:204;        and    -   (c) HVR-L3 comprises the amino acid sequence of SEQ ID NO:205.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising three heavy chain hypervariableregions (HVR-H1, HVR-H2, and HVR-H3), wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:200;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:201;        and    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:202.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, and/or threelight chain hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-L1 comprises the amino acid sequence of SEQ ID NO:203;    -   (b) HVR-L2 comprises the amino acid sequence of SEQ ID NO:204;        and    -   (c) HVR-L3 comprises the amino acid sequence of SEQ ID NO:205.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, and/or threeheavy chain hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:200;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:201;        and    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:202.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain variable region and a lightchain variable region, wherein the heavy chain variable region comprisesan amino acid sequence selected from the group consisting of SEQ IDNOs:154 and 158, and the light chain variable region comprises the aminoacid sequence of SEQ ID NO:156.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a light chain variable region comprising theamino acid sequence of SEQ ID NO:156.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain variable region comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:154 and 158.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain and a light chain, wherein theheavy chain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs:153 and 157, and the light chain comprises theamino acid sequence of SEQ ID NO:155.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a light chain comprising the amino acidsequence of SEQ ID NO:155.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:153 and 157.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises three heavy chain hypervariable regions(HVR-H1, HVR-H2, and HVR-H3) and three light chain hypervariable regions(HVR-L1, HVR-L2, and HVR-L3), wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:206;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:207;    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:208;    -   (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO:209;    -   (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO:210;        and    -   (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO:211.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, three, four,five and/or six hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:206;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:207;    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:208;    -   (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO:209;    -   (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO:210;        and    -   (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO:211.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising three light chain hypervariableregions (HVR-L1, HVR-L2, and LVR-L3), wherein:

-   -   (a) HVR-L1 comprises the amino acid sequence of SEQ ID NO:209;    -   (b) HVR-L2 comprises the amino acid sequence of SEQ ID NO:210;        and    -   (c) HVR-L3 comprises the amino acid sequence of SEQ ID NO:211.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising three heavy chain hypervariableregions (HVR-H1, HVR-H2, and HVR-H3), wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:206;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:207;        and    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:208.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, and/or threelight chain hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-L1 comprises the amino acid sequence of SEQ ID NO:209;    -   (b) HVR-L2 comprises the amino acid sequence of SEQ ID NO:210;        and    -   (c) HVR-L3 comprises the amino acid sequence of SEQ ID NO:211.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, and/or threeheavy chain hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:206;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:207;        and    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:208.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain variable region and a lightchain variable region, wherein the heavy chain variable region comprisesthe amino acid sequence of SEQ ID NO:160, and the light chain variableregion comprises the amino acid sequence of SEQ ID NO:162.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a light chain variable region comprising theamino acid sequence of SEQ ID NO:162.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain variable region comprises theamino acid sequence of SEQ ID NO: 160.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain and a light chain, wherein theheavy chain comprises the amino acid sequence of SEQ ID NO:159, and thelight chain comprises the amino acid sequence of SEQ ID NO:161.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a light chain comprising the amino acidsequence of SEQ ID NO:161.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain comprising the amino acidsequence of SEQ ID NO:159.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises three heavy chain hypervariable regions(HVR-H1, HVR-H2, and HVR-H3) and three light chain hypervariable regions(HVR-L1, HVR-L2, and HVR-L3), wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:212;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:213;    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:214;    -   (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO:215;    -   (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO:216;        and    -   (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO:217.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, three, four,five and/or six hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:212;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:213;    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:214;    -   (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO:215;    -   (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO:216;        and    -   (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO:217.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising three light chain hypervariableregions (HVR-L1, HVR-L2, and LVR-L3), wherein:

-   -   (a) HVR-L1 comprises the amino acid sequence of SEQ ID NO:215;    -   (b) HVR-L2 comprises the amino acid sequence of SEQ ID NO:216;        and    -   (c) HVR-L3 comprises the amino acid sequence of SEQ ID NO:217.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising three heavy chain hypervariableregions (HVR-H1, HVR-H2, and HVR-H3), wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:212;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:213;        and    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:214.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, and/or threelight chain hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-L1 comprises the amino acid sequence of SEQ ID NO:215;    -   (b) HVR-L2 comprises the amino acid sequence of SEQ ID NO:216;        and    -   (c) HVR-L3 comprises the amino acid sequence of SEQ ID NO:217.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, and/or threeheavy chain hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:212;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:213;        and    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:214.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain variable region and a lightchain variable region, wherein the heavy chain variable region comprisesthe amino acid sequence of SEQ ID NO:164, and the light chain variableregion comprises the amino acid sequence of SEQ ID NO:166.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a light chain variable region comprising theamino acid sequence of SEQ ID NO:166.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain variable region comprises theamino acid sequence of SEQ ID NO: 164.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain and a light chain, wherein theheavy chain comprises the amino acid sequence of SEQ ID NO:163, and thelight chain comprises the amino acid sequence of SEQ ID NO:165.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a light chain comprising the amino acidsequence of SEQ ID NO:165.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain comprising the amino acidsequence of SEQ ID NO:163.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises three heavy chain hypervariable regions(HVR-H1, HVR-H2, and HVR-H3) and three light chain hypervariable regions(HVR-L1, HVR-L2, and HVR-L3), wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:218;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:219;    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:220;    -   (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO:221;    -   (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO:222;        and    -   (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO:223.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, three, four,five and/or six hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:218;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:219;    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:220;    -   (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO:221;    -   (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO:222;        and    -   (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO:223.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising three light chain hypervariableregions (HVR-L1, HVR-L2, and LVR-L3), wherein:

-   -   (a) HVR-L1 comprises the amino acid sequence of SEQ ID NO:221;    -   (b) HVR-L2 comprises the amino acid sequence of SEQ ID NO:222;        and    -   (c) HVR-L3 comprises the amino acid sequence of SEQ ID NO:223.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising three heavy chain hypervariableregions (HVR-H1, HVR-H2, and HVR-H3), wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:218;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:219;        and    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:220.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, and/or threelight chain hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-L1 comprises the amino acid sequence of SEQ ID NO:221;    -   (b) HVR-L2 comprises the amino acid sequence of SEQ ID NO:222;        and    -   (c) HVR-L3 comprises the amino acid sequence of SEQ ID NO:223.

In some embodiments, the invention provides an isolatedanti-hemagglutinin antibody comprising: at least one, two, and/or threeheavy chain hypervariable region (HVR) sequences, wherein:

-   -   (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO:218;    -   (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO:219;        and    -   (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:220.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain variable region and a lightchain variable region, wherein the heavy chain variable region comprisesthe amino acid sequence of SEQ ID NO:168, and the light chain variableregion comprises the amino acid sequence of SEQ ID NO:170.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a light chain variable region comprising theamino acid sequence of SEQ ID NO:170.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain variable region comprises theamino acid sequence of SEQ ID NO: 168.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain and a light chain, wherein theheavy chain comprises the amino acid sequence of SEQ ID NO:167, and thelight chain comprises the amino acid sequence of SEQ ID NO:169.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a light chain comprising the amino acidsequence of SEQ ID NO:169.

In some embodiments, an isolated anti-hemagglutinin antibody of thepresent invention comprises a heavy chain comprising the amino acidsequence of SEQ ID NO:167.

The invention also provides isolated nucleic acids encoding ananti-hemagglutinin antibody of the present invention. The invention alsoprovides vectors comprising a nucleic acid encoding ananti-hemagglutinin antibody of the present invention. The invention alsoprovides host cells comprising a nucleic acid or a vector of the presentinvention. A vector can be of any type, for example, a recombinantvector such as an expression vector. Any of a variety of host cells canbe used. In one embodiment, a host cell is a prokaryotic cell, forexample, E. coli. In another embodiment, a host cell is a eukaryoticcell, for example, a mammalian cell, such as a Chinese Hamster Ovary(CHO) cell.

The invention further provides a method of producing ananti-hemagglutinin antibody of the present invention. For example, theinvention provides methods for making an anti-hemagglutinin antibody(which, as defined herein, includes full length antibody and fragmentsthereof), the method comprising expressing in a suitable host cell arecombinant vector of the invention encoding the anti-hemagglutininantibody or fragments thereof so that the antibody or fragments thereofare produced. In some embodiments, the method comprises culturing a hostcell comprising nucleic acid encoding an anti-hemagglutinin antibody ofthe present invention (or fragments thereof) so that the nucleic acid isexpressed. The method may further comprise recovering theanti-hemagglutinin antibody or fragments thereof from the host cellculture or the host cell culture medium.

The invention also provides a pharmaceutical formulation comprising ananti-hemagglutinin antibody of the present invention and apharmaceutically acceptable carrier. The pharmaceutical formulation mayfurther comprise an additional therapeutic agent (e.g., a neuraminidaseinhibitor, such as oseltamivir or zanamivir; another antibody, such asanother anti-hemagglutinin antibody or an anti-M2 antibody; etc).

The invention also provides compositions comprising ananti-hemagglutinin antibody of the present invention. The compositionmay further comprise an additional therapeutic agent (e.g., aneuraminidase inhibitor, such as oseltamivir or zanamivir; anotherantibody, such as another anti-hemagglutinin antibody or an anti-M2antibody; etc).

The invention also provides a composition comprising ananti-hemagglutinin antibody of the present invention for use inpreventing influenza A virus infection. In some embodiments, theinvention provides a pharmaceutical composition comprising ananti-hemagglutinin antibody of the present invention for use inpreventing influenza A virus infection. The invention further provides acomposition comprising an anti-hemagglutinin antibody of the presentinvention for use in treating influenza A virus infection. In someembodiments, the invention provides a pharmaceutical compositioncomprising an anti-hemagglutinin antibody of the present invention foruse in treating influenza A virus infection. The invention furtherprovides a composition comprising an anti-hemagglutinin antibody of thepresent invention for use in inhibiting influenza A virus infection. Insome embodiments, the invention provides a pharmaceutical compositioncomprising an anti-hemagglutinin antibody of the present invention foruse in inhibiting influenza A virus infection.

Compositions comprising an anti-hemagglutinin antibody of the presentinvention may also be used in the manufacture of a medicament. Themedicament may be for use in the inhibition, treatment, or prevention ofinfluenza A virus infection. In certain embodiments, the medicament mayfurther comprise an additional therapeutic agent (e.g., a neuraminidaseinhibitor, such as oseltamivir or zanamivir; another antibody, such asanother anti-hemagglutinin antibody or an anti-M2 antibody; etc).

The invention also provides a method for inhibiting influenza A virusinfection, the method comprising administering to a patient in needthereof an effective amount of a composition comprising ananti-hemagglutinin antibody of the present invention, thereby inhibitinginfluenza A virus infection. The invention also provides a method fortreating influenza A virus infection, the method comprisingadministering to a patient in need thereof an effective amount of acomposition comprising an anti-hemagglutinin antibody of the presentinvention, thereby treating influenza A virus infection. The inventionalso provides a method for preventing influenza A virus infection, themethod comprising administering to a patient in need thereof aneffective amount of a composition comprising an anti-hemagglutininantibody of the present invention, thereby preventing influenza A virusinfection.

The invention also provides a method for inhibiting, treating, orpreventing influenza A virus infection, the method comprisingadministering to a patient in need thereof an effective amount of acomposition comprising an anti-hemagglutinin antibody of the presentinvention, and administering to the patient an effective amount of anadditional therapeutic agent, thereby inhibiting, treating, orpreventing influenza A virus infection. In some embodiments, theadditional therapeutic agent is a neuraminidase inhibitor, such asoseltamivir or zanamivir.

In other embodiments, the additional therapeutic agent is anotheranti-hemagglutinin antibody. In yet other embodiments, the additionaltherapeutic agent is an anti-M2 antibody. In various aspects of suchcombination treatments, the therapeutic agents are administered at aboutthe same time, are administered together, or are administeredsequentially or consecutively. In particular embodiments, ananti-neuraminidase inhibitor is administered prior to the administrationof an anti-hemagglutinin antibody of the present invention.

In another aspect, the invention provides use of an anti-hemagglutininantibody of the present invention in the manufacture of a medicament.The medicament may be for use in the inhibition, treatment, orprevention of influenza A virus infection. In certain embodiments, themedicament may further comprise an additional therapeutic agent (e.g., aneuraminidase inhibitor, such as oseltamivir or zanamivir; anotherantibody, such as another anti-hemagglutinin antibody or an anti-M2antibody; etc).

In another aspect, the invention provides use of a nucleic acid of theinvention in the manufacture of a medicament. The medicament may be foruse in the inhibition, treatment, or prevention of influenza A virusinfection. In certain embodiments, the medicament may further comprisean additional therapeutic agent (e.g., a neuraminidase inhibitor, suchas oseltamivir or zanamivir; another antibody, such as anotheranti-hemagglutinin antibody or an anti-M2 antibody; etc).

In another aspect, the invention provides use of an expression vector ofthe invention in the manufacture of a medicament. The medicament may befor use in the inhibition, treatment, or prevention of influenza A virusinfection. In certain embodiments, the medicament may further comprisean additional therapeutic agent (e.g., a neuraminidase inhibitor, suchas oseltamivir or zanamivir; another antibody, such as anotheranti-hemagglutinin antibody or an anti-M2 antibody; etc).

In another aspect, the invention provides use of a host cell of theinvention in the manufacture of a medicament. The medicament may be foruse in the inhibition, treatment, or prevention of influenza A virusinfection. In certain embodiments, the medicament may further comprisean additional therapeutic agent (e.g., a neuraminidase inhibitor, suchas oseltamivir or zanamivir; another antibody, such as anotheranti-hemagglutinin antibody or an anti-M2 antibody; etc).

In another aspect, the invention provides use of an article ofmanufacture of the invention in the manufacture of a medicament. Themedicament may be for use in the inhibition, treatment, or prevention ofinfluenza A virus infection. In certain embodiments, the medicament mayfurther comprise an additional therapeutic agent (e.g., a neuraminidaseinhibitor, such as oseltamivir or zanamivir; another antibody, such asanother anti-hemagglutinin antibody or an anti-M2 antibody; etc).

In another aspect, the invention provides use of a kit of the inventionin the manufacture of a medicament. The medicament may be for use in theinhibition, treatment, or prevention of influenza A virus infection. Incertain embodiments, the medicament may further comprise an additionaltherapeutic agent (e.g., a neuraminidase inhibitor, such as oseltamiviror zanamivir; another antibody, such as another anti-hemagglutininantibody or an anti-M2 antibody; etc).

In various aspects, an anti-hemagglutinin antibody of the presentinvention binds hemagglutinin. In some aspects, an anti-hemagglutininantibody of the present invention binds Group1 hemagglutinin, bindsGroup2 hemagglutinin, or binds Group1 and Group2 hemagglutinin. In otheraspects, an anti-hemagglutinin antibody of the present invention bindshemagglutinin and neutralizes influenza A virus. In some embodiments, ananti-hemagglutinin antibody of the present invention neutralizesinfluenza A virus in vitro, in vivo, or in vitro and in vivo.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B sets forth data showing FACS analysis ofanti-hemagglutinin-positive (hemagglutinin H3+ and hemagglutinin H1+)plasmablasts from day 7 post-vaccinated human peripheral bloodmononuclear cells (PBMCs) prior to SCID/beige mouse enrichment (FIG. 1A)and day 8 post-intrasplenic implantation after SCID/beige mouseenrichment with and without antigen premix (FIG. 1B) in the upper andlower panels, respectively.

FIG. 2 sets forth data showing analysis of splenocytes obtained from day8 post-intrasplenic implantation of PBMCs from individual SCID/beigemice with no PBMC/antigen premix (circles) and with PBMC/antigen premix(squares), as percent hemagglutinin (H1)⁺/CD38^(high) plasmablasts. Therectangle indicates mice that presented hemagglutinin H1⁺ plasmablasts.

FIG. 3 sets for data showing in vitro neutralization of variousinfluenza A Group1 and Group2 virus strains by anti-hemagglutininantibodies of the present invention.

FIGS. 4A and 4B set forth data showing in vitro neutralization ofvarious influenza A Group1 (FIG. 4A) and Group2 (FIG. 4B) virus strainsby monoclonal antibody 39.29 NWPP (“NWPP” disclosed as SEQ ID NO: 177).

FIGS. 5A and 5B set forth data showing in vitro neutralization ofvarious influenza A Group1 (FIG. 5A) and Group2 (FIG. 5B) virus strainsby monoclonal antibody 81.39 SVSH-NYP (“SVSH” disclosed as SEQ ID NO:171).

FIG. 6 sets forth data showing in vitro neutralization of variousinfluenza A Group1 virus strains by monoclonal antibody 39.18 B11.

FIG. 7 sets forth data showing in vitro neutralization of variousinfluenza A Group1 and Group2 virus strains by monoclonal antibody36.89.

FIG. 8 sets forth data showing in vitro neutralization of variousinfluenza A Group1 and Group2 virus strains by monoclonal antibody mAb901F3.

FIG. 9 sets forth data showing in vitro neutralization of variousinfluenza A Group 1 and Group2 virus strains by monoclonal antibodymAb23 06C2.

FIG. 10 sets forth data showing in vitro neutralization of anhemagglutinin H5-expressing pseudovirus by monoclonal antibody 39.29NCv1.

FIG. 11 sets forth data showing in vitro neutralization of an H7N7equine influenza virus by monoclonal antibody 39.29 NWPP (“NWPP”disclosed as SEQ ID NO: 177).

FIGS. 12A, 12B, 12C, and 12D set forth data showing percent survival ofmice infected with various influenza A virus strains (A/PR/8/1934 (PR8),FIG. 12A; A/Port Chalmers/1/1973 (PC73), FIG. 12B; A/Hong Kong/1/1968(HK68), FIG. 12C); and A/Aichi/2/1968 (Aichi68), FIG. 12D) andadministered various amounts of monoclonal antibody 39.29 NWPP (“NWPP”disclosed as SEQ ID NO: 177).

FIG. 13 sets forth data showing percent survival of mice infected withA/PR/8/1934 influenza A virus and administered various amounts ofmonoclonal antibody 39.29 NCv1.

FIG. 14 sets forth data showing percent survival of mice infected withA/Hong Kong/1/1968 influenza A virus (an influenza A virus having a highIC50) and administered various amounts of monoclonal antibody 39.29NCv1.

FIG. 15 sets forth data showing percent survival of mice infected withA/Port Chalmers/1/1973 influenza A virus and administered variousamounts of monoclonal antibody 39.29 NCv1.

FIG. 16 sets forth data showing percent survival of mice infected withA/Aichi/2/1968 influenza A virus and administered various amounts ofmonoclonal antibody 39.29 NCv1.

FIG. 17 sets forth data comparing percent survival of mice infected withinfluenza A virus strain A/PR/8/1934 and administered a 50:50 mixture ofmonoclonal antibody 39.29 D8C2 and monoclonal antibody 39.29 NWPP(“NWPP” disclosed as SEQ ID NO: 177) or oseltamivir (Tamiflu®).

FIG. 18 sets forth data showing comparing percent survival of miceinfected with influenza A virus strain A/PR/8/1934 and administeredmonoclonal antibody 39.29 NWPP (“NWPP” disclosed as SEQ ID NO: 177),oseltamivir (Tamiflu®), or a combination of monoclonal antibody 39.29NWPP (“NWPP” disclosed as SEQ ID NO: 177) and oseltamivir.

FIGS. 19A and 19B set for data comparing percent survival of ferretsinfected with influenza A virus strain A/Vietnam/1203/04 (H5N1) andadministered monoclonal antibody 39.29 D8C2 (FIG. 19A), monoclonalantibody 81.39 B1C1 (FIG. 19B), or oseltamivir (Tamiflu®) at 48 hours or72 hours post-infection.

FIG. 20 shows an amino acid sequence alignment of hemagglutinin aminoacid sequences from hemagglutinin H1, H2, H3, H5 and H7, showinghemagglutinin contact residues (shaded) of monoclonal antibody 39.29NCv1and the hemagglutinin binding epitope.

FIGS. 21A and 21B set forth data from competition ELISA experiments ofvarious monoclonal antibodies of the present invention competing withbinding of biotin-labeled monoclonal antibody 39.29 to hemagglutinin H1from A/NWS/1933 (FIG. 21A) and hemagglutinin H3 from A/HK/8/1968 (FIG.21B).

FIGS. 22A and 22B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 81.39 B1C1 (SEQ ID NOs:113 and 111, respectively) with theimmunoglobulin kappa variable 3-15*01 germ-line (IGKV3-15*01) and theimmunoglobulin heavy chain variable 3-30*01 germ-line (IGHV3-30*01) (SEQID NOs:236 and 237, respectively). The amino acids are numbers accordingto Kabat numbering. The Kabat, Chothia, and Contact CDRs are indicated.

FIGS. 23A and 23B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 81.39 SVSH-NYP (“SVSH” disclosed as SEQ ID NO: 171) (SEQ IDNOs:117 and 115, respectively) with immunoglobulin kappa variable3-15*01 germ-line (IGKV3-15*01) and the immunoglobulin heavy chainvariable 3-30*01 germ-line (IGHV3-30*01) (SEQ ID NOs:236 and 237,respectively). The amino acids are numbers according to Kabat numbering.The Kabat, Chothia, and Contact CDRs are indicated.

FIGS. 24A and 24B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 81.39 B1F1 (SEQ ID NOs:119 and 111, respectively) with theimmunoglobulin kappa variable 3-15*01 germ-line (IGKV3-15*01) and theimmunoglobulin heavy chain variable 3-30*01 germ-line (IGHV3-30*01) (SEQID NOs:236 and 237, respectively). The amino acids are numbers accordingto Kabat numbering. The Kabat, Chothia, and Contact CDRs are indicated.

FIGS. 25A and 25B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 81.39 SVDS (“SVDS” disclosed as SEQ ID NO: 172) (SEQ ID NOs:113and 115, respectively) with the immunoglobulin kappa variable 3-15*01germ-line (IGKV3-15*01) and the immunoglobulin heavy chain variable3-30*01 germ-line (IGHV3-30*01) (SEQ ID NOs:236 and 237, respectively).The amino acids are numbers according to Kabat numbering. The Kabat,Chothia, and Contact CDRs are indicated.

FIGS. 26A and 26B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 81.39 SVSS (“SVSS” disclosed as SEQ ID NO: 173) (SEQ ID NOs:122and 115, respectively) with the immunoglobulin kappa variable 3-15*01germ-line (IGKV3-15*01) and the immunoglobulin heavy chain variable3-30*01 germ-line (IGHV3-30*01) (SEQ ID NOs:236 and 237, respectively).The amino acids are numbers according to Kabat numbering. The Kabat,Chothia, and Contact CDRs are indicated.

FIGS. 27A and 27B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 81.39 SVDH (“SVDH” disclosed as SEQ ID NO: 174) (SEQ ID NOs:124and 115, respectively) with the immunoglobulin kappa variable 3-15*01germ-line (IGKV3-15*01) and the immunoglobulin heavy chain variable3-30*01 germ-line (IGHV3-30*01) (SEQ ID NOs:236 and 237, respectively).The amino acids are numbers according to Kabat numbering. The Kabat,Chothia, and Contact CDRs are indicated.

FIGS. 28A and 28B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of mAb 81.39SVSH (“SVSH” disclosed as SEQ ID NO: 171) (SEQ ID NOs:126 and 115,respectively) with the immunoglobulin kappa variable 3-15*01 germ-line(IGKV3-15*01) and the immunoglobulin heavy chain variable 3-30*01germ-line (IGHV3-30*01) (SEQ ID NOs:236 and 237, respectively). Theamino acids are numbers according to Kabat numbering. The Kabat,Chothia, and Contact CDRs are indicated.

FIGS. 29A and 29B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 81.39 SVSH.NFP (“SVSH” disclosed as SEQ ID NO: 171) (SEQ IDNOs:128 and 115, respectively) with the immunoglobulin kappa variable3-15*01 germ-line (IGKV3-15*01) and the immunoglobulin heavy chainvariable 3-30*01 germ-line (IGHV3-30*01) (SEQ ID NOs:236 and 237,respectively). The amino acids are numbers according to Kabat numbering.The Kabat, Chothia, and Contact CDRs are indicated.

FIGS. 30A and 30B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 81.39 SVDS.F (“SVDS” disclosed as SEQ ID NO: 172) (SEQ IDNOs:130 and 115, respectively) with the immunoglobulin kappa variable3-15*01 germ-line (IGKV3-15*01) and the immunoglobulin heavy chainvariable 3-30*01 germ-line (IGHV3-30*01) (SEQ ID NOs:236 and 237,respectively). The amino acids are numbers according to Kabat numbering.The Kabat, Chothia, and Contact CDRs are indicated.

FIGS. 31A and 31B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 81.39 SVDS.Y (“SVDS” disclosed as SEQ ID NO: 172) (SEQ IDNOs:132 and 115, respectively) with the immunoglobulin kappa variable3-15*01 germ-line (IGKV3-15*01) and the immunoglobulin heavy chainvariable 3-30*01 germ-line (IGHV3-30*01) (SEQ ID NOs:236 and 237,respectively). The amino acids are numbers according to Kabat numbering.The Kabat, Chothia, and Contact CDRs are indicated.

FIGS. 32A and 32B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 39.29 D2C4 (SEQ ID NOs:136 and 134, respectively) with theimmunoglobulin kappa variable 3-15*01 germ-line (IGKV3-15*01) and theimmunoglobulin heavy chain variable 3-30*01 germ-line (IGHV3-30*01) (SEQID NOs:236 and 245, respectively). The amino acids are numbers accordingto Kabat numbering. The Kabat, Chothia, and Contact CDRs are indicated.

FIGS. 33A and 33B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 39.29 D8C2 (SEQ ID NOs:140 and 138, respectively) with theimmunoglobulin kappa variable 3-15*01 germ-line (IGKV3-15*01) and theimmunoglobulin heavy chain variable 3-30*01 germ-line (IGHV3-30*01) (SEQID NOs:236 and 245, respectively). The amino acids are numbers accordingto Kabat numbering. The Kabat, Chothia, and Contact CDRs are indicated.

FIGS. 34A and 34B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 39.29 NCv1 (SEQ ID NOs:144 and 142, respectively) with theimmunoglobulin kappa variable 3-15*01 germ-line (IGKV3-15*01) and theimmunoglobulin heavy chain variable 3-30*01 germ-line (IGHV3-30*01) (SEQID NOs:236 and 245, respectively). The amino acids are numbers accordingto Kabat numbering. The Kabat, Chothia, and Contact CDRs are indicated.

FIGS. 35A and 35B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 39.29 D8E7 (SEQ ID NOs:146 and 138, respectively) with theimmunoglobulin kappa variable 3-15*01 germ-line (IGKV3-15*01) and theimmunoglobulin heavy chain variable 3-30*01 germ-line (IGHV3-30*01) (SEQID NOs:236 and 245, respectively). The amino acids are numbers accordingto Kabat numbering. The Kabat, Chothia, and Contact CDRs are indicated.

FIGS. 36A and 36B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 39.29 NFPP (“NFPP” disclosed as SEQ ID NO: 175) (SEQ ID NOs:150and 148, respectively) with the immunoglobulin kappa variable 3-15*01germ-line (IGKV3-15*01) and the immunoglobulin heavy chain variable3-30*01 germ-line (IGHV3-30*01) (SEQ ID NOs:236 and 245, respectively).The amino acids are numbers according to Kabat numbering. The Kabat,Chothia, and Contact CDRs are indicated.

FIGS. 37A and 37B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 39.29 NYPP (“NYPP” disclosed as SEQ ID NO: 176) (SEQ ID NOs:152and 148, respectively) with the immunoglobulin kappa variable 3-15*01germ-line (IGKV3-15*01) and the immunoglobulin heavy chain variable3-30*01 germ-line (IGHV3-30*01) (SEQ ID NOs:236 and 245, respectively).The amino acids are numbers according to Kabat numbering. The Kabat,Chothia, and Contact CDRs are indicated.

FIGS. 38A and 38B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 39.29 NWPP (“NWPP” disclosed as SEQ ID NO: 177) (SEQ ID NOs:235and 234, respectively) with the immunoglobulin kappa variable 3-15*01germ-line (IGKV3-15*01) and the immunoglobulin heavy chain variable3-30*01 germ-line (IGHV3-30*01) (SEQ ID NOs:236 and 245, respectively).The amino acids are numbers according to Kabat numbering. The Kabat,Chothia, and Contact CDRs are indicated.

FIGS. 39A and 39B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 39.18 B11 (SEQ ID NOs:156 and 154, respectively) with theimmunoglobulin kappa variable 3-15*01 germ-line (IGKV3-15*01) and theimmunoglobulin heavy chain variable 1-69*01 germ-line (IGHV1-69*01) (SEQID NOs:236 and 238, respectively). The amino acids are numbers accordingto Kabat numbering. The Kabat, Chothia, and Contact CDRs are indicated.

FIGS. 40A and 40B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 39.18 E12 (SEQ ID NOs:156 and 158, respectively) with theimmunoglobulin kappa variable 3-15*01 germ-line (IGKV3-15*01) and theimmunoglobulin heavy chain variable 1-69*01 germ-line (IGHV1-69*01) (SEQID NOs:236 and 238, respectively). The amino acids are numbers accordingto Kabat numbering. The Kabat, Chothia, and Contact CDRs are indicated.

FIGS. 41A and 41B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 36.89 (SEQ ID NOs:162 and 160, respectively) with theimmunoglobulin kappa variable 1-5*03 germ-line (IGKV1-5*03) and theimmunoglobulin heavy chain variable 1-18*01 germ-line (IGHV1-18*01) (SEQID NOs:239 and 240, respectively). The amino acids are numbers accordingto Kabat numbering. The Kabat, Chothia, and Contact CDRs are indicated.

FIGS. 42A and 42B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 9.01F3 (SEQ ID NOs:166 and 164, respectively) with theimmunoglobulin light variable 1-44*01 germ-line (IGKV1-44*01) and theimmunoglobulin heavy chain variable 1-2*02*01 germ-line (IGHV1-2*02)(SEQ ID NOs:241 and 242, respectively). The amino acids are numbersaccording to Kabat numbering. The Kabat, Chothia, and Contact CDRs areindicated.

FIGS. 43A and 43B show an amino acid sequence alignment of the lightchain variable region and the heavy chain variable region of monoclonalantibody 23.06C2 (SEQ ID NOs:170 and 168, respectively) with theimmunoglobulin kappa variable 2-30*01 germ-line (IGKV2-30*01) and theimmunoglobulin heavy chain variable 4-39*01 germ-line (IGHV4-39*01) (SEQID NOs:243 and 244, respectively). The amino acids are numbers accordingto Kabat numbering. The Kabat, Chothia, and Contact CDRs are indicated.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-hemagglutinin antibody” and “an antibody that binds tohemagglutinin” refer to an antibody that binds hemagglutinin withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting hemagglutinin, including targetinghemagglutinin of influenza virus. In one embodiment, the extent ofbinding of an anti-hemagglutinin antibody to an unrelated,non-hemagglutinin protein is less than about 10% of the binding of theantibody to hemagglutinin as measured, e.g., by a radioimmunoassay(RIA). In certain embodiments, an antibody that binds to hemagglutininhas a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10⁻⁸ M or less, e.g., from 10⁻⁸ M to10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certain embodiments, ananti-hemagglutinin antibody binds to an epitope of hemagglutinin that isconserved among hemagglutinin from different strains, subtypes, andisolates of influenza A viruses.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. An antibody fragment alsorefers to a molecule other than an intact antibody that comprises aportion of an intact antibody that binds hemagglutinin and neutralizesinfluenza A virus. Examples of antibody fragments include but are notlimited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies; linearantibodies; single-chain antibody molecules (e.g., scFv); andmultispecific antibodies formed from antibody fragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is an antibody which possesses an amino acid sequencewhich corresponds to that of an antibody produced by a human or a humancell or derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH(H1, H2, H3), and three in the VL (L1,L2, L3). Exemplary HVRs herein include:

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat et al., Sequences of Proteins of Immunological Interest,        5th Ed. Public Health Service, National Institutes of Health,        Bethesda, Md. (1991));    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)); and    -   (d) combinations of (a), (b), and/or (c), including HVR amino        acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),        26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102        (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-hemagglutinin antibody” refersto one or more nucleic acid molecules encoding antibody heavy and lightchains (or fragments thereof), including such nucleic acid molecule(s)in a single vector or separate vectors, and such nucleic acidmolecule(s) present at one or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “hemagglutinin,” as used herein, refers to any nativehemagglutinin from any influenza virus source, unless otherwiseindicated. The term encompasses “full-length,” unprocessed hemagglutininas well as any form of hemagglutinin that results from processing in aninfluenza virus or an influenza virus-infected cell. The term alsoencompasses naturally occurring variants of hemagglutinin, e.g., splicevariants or allelic variants. The amino acid sequences of exemplaryhemagglutinin proteins from various influenza A virus strains are shownin SEQ ID NOs:225 (H2 from A/Japan/305/1957), 226 (H3 fromA/Perth/16/2009), 227 (H5 from A/Vietnam/1203/2004), 228 (H7 fromA/chicken/NSW/1/1997), 229 (H1 from A/California/07/2009), 230 (H1 fromA/NSW/1933), 231 (H3 from A/Hong Kong/8/1968), 232 (H7 fromA/Netherlands/219/2003), and 233 (A/South Carolina/1918).

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease (e.g., preventingoccurrence or recurrence of influenza A virus infection), reduction(e.g., reducing) or alleviation of symptoms, diminishment of any director indirect pathological consequences of the disease, decreasing therate of disease progression, amelioration or palliation of the diseasestate, and remission or improved prognosis. In some embodiments,antibodies of the invention are used to delay development of a diseaseor to slow the progression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

II. Compositions and Methods

In one aspect, the invention is based, in part, on anti-hemagglutininantibodies and uses thereof. In certain embodiments, antibodies thatbind to hemagglutinin are provided. Antibodies of the invention areuseful, e.g., for the diagnosis, treatment, or prevention of influenza Avirus infection.

A. Exemplary Anti-Hemagglutinin Antibodies

In one aspect, the invention provides isolated antibodies that bind tohemagglutinin. In certain embodiments, an anti-hemagglutinin antibody ofthe present invention binds hemagglutinin, binds Group1 hemagglutinins,binds Group2 hemagglutinins, or binds Group1 and Group2 hemagglutinins.In other embodiments, an anti-hemagglutinin antibody of the presentinvention neutralizes influenza A virus in vitro. In other embodiments,an anti-hemagglutinin antibody of the present invention neutralizesinfluenza A virus in vivo. In yet other embodiments, ananti-hemagglutinin antibody of the present invention reduces influenza Avirus infection, prevents influenza A virus infection, inhibitsinfluenza A virus infection, or treats influenza A virus infection. Insome embodiments, an anti-hemagglutinin antibody of the presentinvention prevents, inhibits, or reduces hemagglutinin-mediated fusionbetween influenza virus membrane and infected cell endosomal membranes(thus preventing, inhibiting, or reducing viral RNA entry into theinfected cell cytoplasm, thus preventing, inhibiting, or reducingfurther propagation of influenza virus infection.)

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:180; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:182; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:188.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:183; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:189.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:182; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:188.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:184; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:188.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:185; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:188.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:183; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:188.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:183; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:190.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:182; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:190.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:186; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:189.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:180 and 181; (d) HVR-L1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:182, 183, 184, 185, and186; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:187; and(f) HVR-L3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:188, 189, and 190.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:179; and (c) HVR-H3comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:180 and 181.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:182, 183, 184, 185, and 186; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (c) HVR-L3comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:188, 189, and 190.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:180; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:182; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:188.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:183; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:189.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:182; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:188.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:184; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:188.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:185; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:188.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:183; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:188.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:183; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:190.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:182; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:190.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:178; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:179; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:181; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:186; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:187; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:189.

In another aspect, the invention provides an antibody comprising a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs:111 and 115.

In another aspect, the invention provides an antibody comprising a lightchain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs:113, 117, 119, 122, 124, 126, 128,130, and 132.

In another aspect, the invention provides an antibody comprising a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs:111 and 115 and a light chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs:113, 117, 119, 122, 124, 126, 128, 130,and 132.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:111 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:113.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:115 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:117.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:111 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:119.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:115 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:113.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:115 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:122.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:115 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:124.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:115 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:126.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:115 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:128.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:115 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:130.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:115 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:132.

In another aspect, the invention provides an antibody comprising a heavychain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:110, 114, and 120.

In another aspect, the invention provides an antibody comprising a lightchain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:112, 116, 118, 121, 123, 125, 127, 129, and131.

In another aspect, the invention provides an antibody comprising a heavychain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:110, 114, and 120, and a light chain comprisingan amino acid sequence selected from the group consisting of SEQ IDNOs:112, 116, 118, 121, 123, 125, 127, 129, and 131.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:110, and a lightchain comprising the amino acid sequence of SEQ ID NO:112.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:114, and a lightchain comprising the amino acid sequence of SEQ ID NO:116.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:110, and a lightchain comprising the amino acid sequence of SEQ ID NO:118.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:114, and a lightchain comprising the amino acid sequence of SEQ ID NO:112.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:120, and a lightchain comprising the amino acid sequence of SEQ ID NO:121.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:114, and a lightchain comprising the amino acid sequence of SEQ ID NO:123.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:114, and a lightchain comprising the amino acid sequence of SEQ ID NO:125.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:114, and a lightchain comprising the amino acid sequence of SEQ ID NO:127.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:114, and a lightchain comprising the amino acid sequence of SEQ ID NO:129.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:114, and a lightchain comprising the amino acid sequence of SEQ ID NO:131.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:191; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:193; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:194; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:195; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:196; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:197.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:192; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:193; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:194; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:195; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:196; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:197.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:191; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:193; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:194; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:195; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:196; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:198.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:191; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:193; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:194; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:195; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:196; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:199.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:191 and 192; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:193; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:194.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:195; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:196; and (c) HVR-L3comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:197, 198, and 199.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:191; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:193; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:194; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:195; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:196; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:197.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:192; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:193; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:194; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:195; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:196; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:197.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:191; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:193; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:194; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:195; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:196; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:198.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:191; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:193; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:194; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:195; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:196; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:199.

In another aspect, the invention provides an antibody comprising a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs:134, 138, 142, 148, and 234.

In another aspect, the invention provides an antibody comprising a lightchain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs:136, 140, 144, 146, 150, 152, and235.

In another aspect, the invention provides an antibody comprising a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs:134, 138, 142, 148, and 234, and alight chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:136, 140, 144, 146, 150, 152,and 235.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:134 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:136.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:138 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:140.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:142 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:144.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:138 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:146.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:148 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:150.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:148 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:152.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:148 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:140.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:234 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:235.

In another aspect, the invention provides an antibody comprising a heavychain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:133, 137, 141, and 147.

In another aspect, the invention provides an antibody comprising a lightchain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:135, 139, 143, 145, 149, and 151.

In another aspect, the invention provides an antibody comprising a heavychain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:133, 137, 141, and 147, and a light chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:135, 139, 143, 145, 149, and 151.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:133, and a lightchain comprising the amino acid sequence of SEQ ID NO:135.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:137, and a lightchain comprising the amino acid sequence of SEQ ID NO:139.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:141, and a lightchain comprising the amino acid sequence of SEQ ID NO:143.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:137, and a lightchain comprising the amino acid sequence of SEQ ID NO:145.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:147, and a lightchain comprising the amino acid sequence of SEQ ID NO:149.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:147, and a lightchain comprising the amino acid sequence of SEQ ID NO:151.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:147, and a lightchain comprising the amino acid sequence of SEQ ID NO:139.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:200; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:201; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:202; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:203; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:204; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:205.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:200; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:201; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:202.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:203; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:204; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:205.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:200; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:201; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:202; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:203; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:204; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:205.

In another aspect, the invention provides an antibody comprising a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs:154 and 158.

In another aspect, the invention provides an antibody comprising a lightchain variable region comprising the amino acid sequence of SEQ IDNO:156.

In another aspect, the invention provides an antibody comprising a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs:154 and 158, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:156.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:154 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:156.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:158 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:156.

In another aspect, the invention provides an antibody comprising a heavychain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:153 and 157.

In another aspect, the invention provides an antibody comprising a lightchain comprising the amino acid sequence of SEQ ID NO:155.

In another aspect, the invention provides an antibody comprising a heavychain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:153 and 157, and a light chain comprising theamino acid sequence of SEQ ID NO:155.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:153, and a lightchain comprising the amino acid sequence of SEQ ID NO:155.

In one embodiment, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:157, and a lightchain comprising the amino acid sequence of SEQ ID NO:155.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:206; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:207; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:208; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:209; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:210; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:211.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:206; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:207; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:208.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:209; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:210; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:211.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:206; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:207; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:208; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:209; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:210; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:211.

In another aspect, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:160.

In another aspect, the invention provides an antibody comprising a lightchain variable region comprising the amino acid sequence of SEQ IDNO:162.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:160 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:162.

In another aspect, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:159.

In another aspect, the invention provides an antibody comprising a lightchain comprising the amino acid sequence of SEQ ID NO:161.

In another aspect, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:159, and a lightchain comprising the amino acid sequence of SEQ ID NO:161.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:212; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:213; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:214; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:215; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:216; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:217.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:212; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:213; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:214.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:215; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:216; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:217.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:212; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:213; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:214; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:215; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:216; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:217.

In another aspect, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:164.

In another aspect, the invention provides an antibody comprising a lightchain variable region comprising the amino acid sequence of SEQ IDNO:166.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:164 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:166.

In another aspect, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:163.

In another aspect, the invention provides an antibody comprising a lightchain comprising the amino acid sequence of SEQ ID NO:165.

In another aspect, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:163, and a lightchain comprising the amino acid sequence of SEQ ID NO:165.

In one aspect, the invention provides an anti-hemagglutinin antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:218; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:219; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:220; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:221; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:222; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:223.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:218; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:219; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:220.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:221; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:222; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:223.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:218; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:219; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:220; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:221; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:222; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:223.

In another aspect, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:168.

In another aspect, the invention provides an antibody comprising a lightchain variable region comprising the amino acid sequence of SEQ IDNO:170.

In one embodiment, the invention provides an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ IDNO:168 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:170.

In another aspect, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:167.

In another aspect, the invention provides an antibody comprising a lightchain comprising the amino acid sequence of SEQ ID NO:169.

In another aspect, the invention provides an antibody comprising a heavychain comprising the amino acid sequence of SEQ ID NO:167, and a lightchain comprising the amino acid sequence of SEQ ID NO:169.

In any of the above embodiments, an anti-hemagglutinin antibody of thepresent invention is humanized. In one embodiment, an anti-hemagglutininantibody comprises HVRs as in any of the above embodiments, and furthercomprises an acceptor human framework, e.g., a human immunoglobulinframework or a human consensus framework.

In another aspect, an anti-hemagglutinin antibody of the presentcomprises a heavy chain variable domain (VH) sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs:111, 115, 134, 138, 142, 148, 154, 158, 160, 164, 168, and234. In certain embodiments, a VH sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-hemagglutininantibody comprising that sequence retains the ability to bind tohemagglutinin. In certain embodiments, a total of 1 to 10 amino acidshave been substituted, inserted and/or deleted in SEQ ID NOs: 111, 115,134, 138, 142, 148, 154, 158, 160, 164, 168, or 234. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs). Optionally, the anti hemagglutininantibody comprises the VH sequence in SEQ ID NO: 111, 115, 134, 138,142, 148, 154, 158, 160, 164, 168, or 234, including post-translationalmodifications of that sequence.

In another aspect, an anti-hemagglutinin antibody is provided, whereinthe antibody comprises a light chain variable domain (VL) having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs:113, 117, 119, 122, 124, 126, 128, 130, 132, 136, 140, 144,146, 150, 152, 156, 162, 166, 170, and 235. In certain embodiments, a VLsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-hemagglutinin antibody comprising that sequence retains the abilityto bind to hemagglutinin. In certain embodiments, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQ IDNOs: 113, 117, 119, 122, 124, 126, 128, 130, 132, 136, 140, 144, 146,150, 152, 156, 162, 166, 170, or 235. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-hemagglutinin antibodycomprises the VL sequence in SEQ ID NOs: 113, 117, 119, 122, 124, 126,128, 130, 132, 136, 140, 144, 146, 150, 152, 156, 162, 166, 170, or 235,including post-translational modifications of that sequence.

In another aspect, an anti-hemagglutinin antibody is provided, whereinthe antibody comprises a VH as in any of the embodiments provided above,and a VL as in any of the embodiments provided above. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NOs: 111, 115,134, 138, 142, 148, 154, 158, 160, 164, 168, or 234, and SEQ ID NOs:113, 117, 119, 122, 124, 126, 128, 130, 132, 136, 140, 144, 146, 150,152, 156, 162, 166, 170, or 235, respectively, includingpost-translational modifications of those sequences.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-hemagglutinin antibody provided herein. Forexample, in certain embodiments, an antibody is provided that binds tothe same epitope as an anti-hemagglutinin antibody comprising a VHsequence of SEQ ID NO:111 and a VL sequence of SEQ ID NO:113; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:117; a VHsequence of SEQ ID NO:111 and a VL sequence of SEQ ID NO:119; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:113; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:122; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:124; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:126; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:128; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:130; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:132; a VHsequence of SEQ ID NO:134 and a VL sequence of SEQ ID NO:136; a VHsequence of SEQ ID NO:138 and a VL sequence of SEQ ID NO:140; a VHsequence of SEQ ID NO:142 and a VL sequence of SEQ ID NO:144; a VHsequence of SEQ ID NO:138 and a VL sequence of SEQ ID NO:146; a VHsequence of SEQ ID NO:148 and a VL sequence of SEQ ID NO:150; a VHsequence of SEQ ID NO:148 and a VL sequence of SEQ ID NO:152; a VHsequence of SEQ ID NO:148 and a VL sequence of SEQ ID NO:140; a VHsequence of SEQ ID NO:234 and a VL sequence of SEQ ID NO:235; a VHsequence of SEQ ID NO:154 and a VL sequence of SEQ ID NO:156; a VHsequence of SEQ ID NO:158 and a VL sequence of SEQ ID NO:156; a VHsequence of SEQ ID NO:160 and a VL sequence of SEQ ID NO:162; a VHsequence of SEQ ID NO:164 and a VL sequence of SEQ ID NO:166; or a VHsequence of SEQ ID NO:168 and a VL sequence of SEQ ID NO:170.

In a further aspect of the invention, an anti-hemagglutinin antibodyaccording to any of the above embodiments is a monoclonal antibody,including a chimeric, humanized, or human antibody. In one embodiment,an anti-hemagglutinin antibody is an antibody fragment, e.g., a Fv, Fab,Fab′, scFv, diabody, or F(ab′)₂ fragment. In another embodiment, theantibody is a full length antibody, e.g., an intact, e.g., IgG1 antibodyor other antibody class or isotype as defined herein.

In a further aspect, an anti-hemagglutinin antibody according to any ofthe above embodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or≦0.001 nM (e.g., 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g.,from 10⁻⁹ M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA). In one embodiment, an RIA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol.Biol. 293:865-881 (1999)). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen antigen are mixed with serial dilutions of a Fabof interest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using a BIACORE® surfaceplasmon resonance assay. For example, an assay using a BIACORE®-2000 ora BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) is performed at 25° C.with immobilized antigen CM5 chips at ˜10 response units (RU). In oneembodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE,Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.,Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g., E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart or using techniques described herein. Human antibodies are describedgenerally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g., a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for hemagglutinin and the other is for any otherantigen. In certain embodiments, bispecific antibodies may bind to twodifferent epitopes of hemagglutinin. Bispecific antibodies may also beused to localize cytotoxic agents to cells which express hemagglutinin.Bispecific antibodies can be prepared as full length antibodies orantibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multispecific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g., US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to hemagglutinin aswell as another, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g., bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or residues that contact antigen,with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al., in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., (2001).) In some embodiments of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g., complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y.et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-hemagglutinin antibody describedherein is provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-hemagglutinin antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-hemagglutinin antibody, nucleicacid encoding an antibody, e.g., as described above, is isolated andinserted into one or more vectors for further cloning and/or expressionin a host cell. Such nucleic acid may be readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.,2003), pp. 245-254, describing expression of antibody fragments in E.coli.) After expression, the antibody may be isolated from the bacterialcell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-hemagglutinin antibodies provided herein may be identified,screened for, or characterized for their physical/chemical propertiesand/or biological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

In another aspect, competition assays may be used to identify anantibody that competes for binding of hemagglutinin with anyanti-hemagglutinin antibody described herein. In certain embodiments,such a competing antibody binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by an anti-hemagglutinin antibodydescribed here (e.g., an anti-hemagglutinin antibody comprising a VHsequence of SEQ ID NO:111 and a VL sequence of SEQ ID NO:113; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:117; a VHsequence of SEQ ID NO:111 and a VL sequence of SEQ ID NO:119; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:113; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:122; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:124; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:126; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:128; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:130; a VHsequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:132; a VHsequence of SEQ ID NO:134 and a VL sequence of SEQ ID NO:136; a VHsequence of SEQ ID NO:138 and a VL sequence of SEQ ID NO:140; a VHsequence of SEQ ID NO:142 and a VL sequence of SEQ ID NO:144; a VHsequence of SEQ ID NO:138 and a VL sequence of SEQ ID NO:146; a VHsequence of SEQ ID NO:148 and a VL sequence of SEQ ID NO:150; a VHsequence of SEQ ID NO:148 and a VL sequence of SEQ ID NO:152; a VHsequence of SEQ ID NO:148 and a VL sequence of SEQ ID NO:140; a VHsequence of SEQ ID NO:234 and a VL sequence of SEQ ID NO:235; a VHsequence of SEQ ID NO:154 and a VL sequence of SEQ ID NO:156; a VHsequence of SEQ ID NO:158 and a VL sequence of SEQ ID NO:156; a VHsequence of SEQ ID NO:160 and a VL sequence of SEQ ID NO:162; a VHsequence of SEQ ID NO:164 and a VL sequence of SEQ ID NO:166; or a VHsequence of SEQ ID NO:168 and a VL sequence of SEQ ID NO:170. Detailedexemplary methods for mapping an epitope to which an antibody binds areprovided in Morris (1996) “Epitope Mapping Protocols,” in Methods inMolecular Biology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized hemagglutinin isincubated in a solution comprising a first labeled antibody that bindsto hemagglutinin and a second unlabeled antibody that is being testedfor its ability to compete with the first antibody for binding tohemagglutinin. The second antibody may be present in a hybridomasupernatant. As a control, immobilized hemagglutinin is incubated in asolution comprising the first labeled antibody but not the secondunlabeled antibody. After incubation under conditions permissive forbinding of the first antibody to hemagglutinin, excess unbound antibodyis removed, and the amount of label associated with immobilizedhemagglutinin is measured. If the amount of label associated withimmobilized hemagglutinin is substantially reduced in the test samplerelative to the control sample, then that indicates that the secondantibody is competing with the first antibody for binding tohemagglutinin. See Harlow and Lane (1988) Antibodies: A LaboratoryManual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-hemagglutininantibodies and fragments thereof having biological activity. Biologicalactivity may include, e.g., specifically binding to influenza A virushemagglutinin, neutralizing influenza A virus, etc. Antibodies andcompositions comprising antibodies or fragments thereof having suchbiological activity in vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the invention is tested for suchbiological activity. See Examples 4, 5, 6, 7, 8, 9, 10, and 13 forexemplary descriptions of such assays.

D. Immunoconjugates

The invention also provides immunoconjugates comprising ananti-hemagglutinin antibody herein conjugated to one or more cytotoxicagents, such as chemotherapeutic agents or drugs, growth inhibitoryagents, toxins (e.g., protein toxins, enzymatically active toxins ofbacterial, fungal, plant, or animal origin, or fragments thereof), orradioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC 1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-hemagglutinin antibodiesprovided herein is useful for detecting the presence of hemagglutinin orinfluenza A virus in a biological sample. The term “detecting” as usedherein encompasses quantitative or qualitative detection. In certainembodiments, a biological sample comprises a cell or tissue, such as,for example, lung, upper respiratory tract, nasal canal, blood, sputum,or comprises a biological sample obtained by nasal or throat swab.

In one embodiment, an anti-hemagglutinin antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of hemagglutinin or influenza A virus in abiological sample is provided. In certain embodiments, the methodcomprises contacting the biological sample with an anti-hemagglutininantibody as described herein under conditions permissive for binding ofthe anti-hemagglutinin antibody to hemagglutinin, and detecting whethera complex is formed between the anti-hemagglutinin antibody andhemagglutinin. Such method may be an in vitro or in vivo method. In oneembodiment, an anti-hemagglutinin antibody is used to select subjectseligible for therapy with an anti-hemagglutinin antibody, e.g., wherehemagglutinin is a biomarker for selection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include influenza A virus infection, including influenza Avirus infection in children, infants, adults, and the elderly.

In certain embodiments, labeled anti-hemagglutinin antibodies areprovided. Labels include, but are not limited to, labels or moietiesthat are detected directly (such as fluorescent, chromophoric,electron-dense, chemiluminescent, and radioactive labels), as well asmoieties, such as enzymes or ligands, that are detected indirectly,e.g., through an enzymatic reaction or molecular interaction. Exemplarylabels include, but are not limited to, the radioisotopes ³²P, ¹⁴C,¹²⁵I, ³H, and ¹³¹I, fluorophores such as rare earth chelates orfluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, luceriferases, e.g., firefly luciferase and bacterialluciferase (U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkalinephosphatase, β-galactosidase, glucoamylase, lysozyme, saccharideoxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-hemagglutinin antibody asdescribed herein are prepared by mixing such antibody having the desireddegree of purity with one or more optional pharmaceutically acceptablecarriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.(1980)), in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent ApplicationPublication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGPis combined with one or more additional glycosaminoglycanases such aschondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide aneuraminidase inhibitor, an anti-hemagglutinin antibody, an anti-M2antibody, etc. Such active ingredients are suitably present incombination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. The formulationsto be used for in vivo administration are generally sterile. Sterilitymay be readily accomplished, e.g., by filtration through sterilefiltration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-hemagglutinin antibodies provided herein may be used intherapeutic methods.

In one aspect, an anti-hemagglutinin antibody for use as a medicament isprovided. In further aspects, an anti-hemagglutinin antibody for use intreating, preventing, or inhibiting influenza A virus infection isprovided. In certain embodiments, an anti-hemagglutinin antibody for usein a method of treatment is provided. In certain embodiments, theinvention provides an anti-hemagglutinin antibody for use in a method oftreating an individual having influenza A virus infection comprisingadministering to the individual an effective amount of theanti-hemagglutinin antibody. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below. Infurther embodiments, the invention provides an anti-hemagglutininantibody for use in preventing, inhibiting, or reducinghemagglutinin-mediated fusion between influenza A virus viral membraneand infected cell endosomal membranes, thus preventing viral RNA entryinto the infected cell cytoplasm and preventing further propagation ofinfection. In certain embodiments, the invention provides ananti-hemagglutinin antibody for use in a method of preventing,inhibiting, or treating influenza A virus infection in an individualcomprising administering to the individual an effective amount of theanti-hemagglutinin antibody to prevent, inhibit, or treat influenza Avirus infection. An “individual” according to any of the aboveembodiments is preferably a human.

In a further aspect, the invention provides for the use of ananti-hemagglutinin antibody in the manufacture or preparation of amedicament. In one embodiment, the medicament is for treatment ofinfluenza A virus infection. In a further embodiment, the medicament isfor use in a method of treating influenza A virus infection comprisingadministering to an individual having influenza A virus infection aneffective amount of the medicament. In one such embodiment, the methodfurther comprises administering to the individual an effective amount ofat least one additional therapeutic agent, e.g., as described below. Ina further embodiment, the medicament is for preventing, inhibiting, orreducing hemagglutinin-mediated fusion between influenza A virus viralmembrane and infected cell endosomal membranes, thus preventing viralRNA entry into the infected cell cytoplasm and preventing furtherpropagation of infection. In a further embodiment, the medicament is foruse in a method of preventing, inhibiting, or treating influenza A virusinfection in an individual comprising administering to the individual anamount effective of the medicament to prevent, inhibit, or reduce,influenza A virus infection. An “individual” according to any of theabove embodiments may be a human.

In a further aspect, the invention provides a method for treatinginfluenza A virus infection. In one embodiment, the method comprisesadministering to an individual having such influenza A virus infectionan effective amount of an anti-hemagglutinin antibody. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent, asdescribed herein. An “individual” according to any of the aboveembodiments may be a human.

The present invention provides anti-hemagglutinin antibodies effectiveat inhibiting, preventing, or treating influenza A virus infection in anindividual (e.g., a subject or a patient). In some aspects, ananti-hemagglutinin antibody of the present invention is effective atprophylactically treating an individual in order to prevent influenza Avirus infection of the individual.

In some aspects, an individual suitable for treatment with ananti-hemagglutinin antibody of the present invention is an individualhaving or suspected having influenza A virus infection. In someembodiments, such individuals include infants, children, adults, and theelderly. In some embodiments, the individual is hospitalized withinfluenza A virus infection. In other embodiments, the individual havinginfluenza A virus infection has one or more co-morbidities, such as, forexample, immunodeficiency, pregnancy, lung disease, heart disease, renaldisease, or co-infection (e.g., a bacterial infection or a viralinfection, such as bacterial or viral pneumonia).

In some aspects, treatment of an individual with an anti-hemagglutininantibody of the present invention reduces influenza A virus infectionseverity, reduces the length of influenza A virus infection, or reducesinfluenza A virus infectivity. In other aspects, treatment of influenzaA virus infection with an anti-hemagglutinin antibody of the presentinvention provides additional benefit, including a reduction in thelength of hospital stay, reduction or prevention of the need forintensive care unit (ICU) use, reduction or prevention of the need forassisted or mechanical ventilation, reduction or prevention of the needfor supplemental oxygen use, and reduction of mortality. In someaspects, the reduction in the length of hospital stay is 1 day, 2 days,3 days, 4 days, 5 days, or longer than 5 days. In some aspects, thereduction in the need for intensive care unit use is 1 day, 2 days, 3days, 4 days, 5 days, or longer than 5 days. In some aspects, thereduction in need for assisted or mechanical ventilation is 1 day, 2days, 3 days, 4 days, 5 days, or longer than 5 days. In some aspects,the reduction in the need for supplemental oxygen is 1 day, 2 days, 3days, 4 days, 5 days, or longer than 5 days. In some aspects, treatmentof an individual with an anti-hemagglutinin antibody of the presentinvention reduces influenza A virus infection disease symptoms, such as,for example, fever, coryza, chills, sore throat, muscle pain, bodyaches, headache, cough, nasal congestion, weakness or fatigue, irritatedor watering eyes, and general discomfort.

In some aspects, treatment of an individual with an anti-hemagglutininantibody of the present invention reduces the time to normalization ofrespiratory function, such as a reduction of time to normalization ofrespiratory rate, or a reduction of time to normalization of oxygensaturation. In some aspects, treatment of an individual with ananti-hemagglutinin antibody of the present invention reduces the time toreturn to normal oxygen saturation, e.g., to an oxygen saturation ofabout 92% or greater, as measured over a 24 hour period withoutsupplemental oxygen administration. In other aspects, treatment of anindividual with an anti-hemagglutinin antibody of the present inventionreduces the time to normalization of vital signs, such as heart rate,blood pressure, respiratory rate, and temperature.

In some aspects, treatment of an individual with an anti-hemagglutininantibody of the present invention improves virologic endpoints, such as,for example, influenza virus titer. Virus titer can be measured byvarious ways known to one of skill in the art, such as, for example,viral area under the curve (AUC), as measured by, for example, qPCR ortissue culture infective does (TCID50). In some aspects, the treatmentresults in greater than or equal to 50% reduction in viral AUC asmeasured by qPCR or TCID50.

In various aspects of the present invention, an anti-hemagglutininantibody provided herein is effective at treating influenza A virusinfection when administered at about 12 hours, at about 24 hours, atabout 36 hours, at about 48 hours, at about 60 hours, at about 72 hours,at about 84 hours, and at about 96 hours after onset of symptoms (e.g.,onset of illness). In other aspects, an anti-hemagglutinin antibodyprovided herein is effective at treating influenza A virus infectionwhen administered between about 24 hours and 48 hours after onset ofsymptoms (e.g., the individual has been symptomatic for between 24 and48 hours), when administered between about 48 hours and 72 hours afteronset of symptoms, or when administered between about 72 hours and 96hours after onset of symptoms. In certain embodiments of the presentinvention, an anti-hemagglutinin antibody of the present invention iseffective at treating or reducing influenza A virus infection andextends the treatment window of current standard of care (e.g.,oseltamivir) beyond 48 hours after onset of symptoms.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-hemagglutinin antibodies provided herein,e.g., for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical formulation comprises any of theanti-hemagglutinin antibodies provided herein and a pharmaceuticallyacceptable carrier. In another embodiment, a pharmaceutical formulationcomprises any of the anti-hemagglutinin antibodies provided herein andat least one additional therapeutic agent, e.g., as described below.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent. In certain embodiments, an additional therapeuticagent is a neuraminidase inhibitor (e.g., zanamivir, oseltamivirphosphate, amantadine, rimantadine), an anti-M2 antibody, ananti-hemagglutinin antibody, etc. In some aspects, treatment of anindividual having influenza A virus infection with an anti-hemagglutininantibody of the present invention co-administered with a neuraminidaseinhibitor provides a synergistic therapeutic effect compared totreatment with either agent alone.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent or agents. In one embodiment, administration of theanti-hemagglutinin antibody and administration of an additionaltherapeutic agent occur within about one month, or within about one,two, or three weeks, within about one, two, three, four, five, or sixdays, or within about one, two, three, four, five, six, eight, ten,twelve, sixteen, twenty, or twenty-four hours of each other.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to about 45 mg/kg (e.g., about 1.0 mg/kgto about 15 mg/kg) of antibody can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. Exemplary dosages of the antibody would be in the range fromabout 1.0 mg/kg to about 45 mg/kg, from about 1.0 mg/kg to about 30mg/kg, from about 1.0 mg/kg to about 15 mg/kg, from about 1.0 mg/kg toabout 10 mg/kg, or from about 1.0 mg/kg to about 5 mg/kg. Thus, one ormore doses of about 1.0 mg/kg, 2.5 mg/kg, 5.0 mg/kg, 10 mg/kg, 15 mg/kg,30 mg/kg, or 45 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.,every day, every two days, every three days, etc. An initial higherloading dose, followed by one or more lower doses may be administered.Dosing can also be at a fixed dose, such as, for example, 200 mg, 400mg, 600 mg, 800 mg, 1000 mg, 1200 mg, 1400 mg, 1500 mg, 1600 mg, 1800mg, 2000 mg, 2200 mg, 2400 mg, 2500 mg, 2600 mg, 2800 mg, 3000 mg, 3200mg, 3400 mg, 3600 mg, etc. The progress of this therapy is easilymonitored by conventional techniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-hemagglutinin antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-hemagglutinin antibody.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1 Identification of Anti-Hemagglutinin Antibodies by PhageDisplay

Construction of Phage Libraries from Influenza Virus Vaccinated HumanDonors

Antibodies directed against influenza A virus hemagglutinin wereidentified using a phage display library constructed from peripheralblood mononuclear cells (PBMCs) isolated from human donors vaccinatedwith the seasonal influenza virus vaccine as follows.

Leukopacs from normal human donors that received the seasonal influenzaFluvirin® vaccine (Novartis Lot #111796P1) 7 days prior to their blooddonation were obtained from Blood Centers of the Pacific (San Francisco,Calif.). PBMCs were isolated from the leukopacs using standardmethodologies. The PBMCs were sorted for CD19⁺/CD20⁻ plasmablast cellsby FACS. RNA from the CD19⁺/CD20⁻ sorted plasmablasts was extractedusing RNeasy purification kit (Qiagen, USA) and cDNA was generated fromthe isolated RNA by reverse transcription using SuperScript® III ReverseTranscriptase (Invitrogen, USA). Human variable heavy (VH), variablekappa (VK), and variable light (VL) genes were PCR amplified from thecDNA using the following back and forward DNA primer mixtures.

VH Back BssHII.HuVH1: (SEQ ID NO: 1)ATCGTTTCATAAGCGCGCCAGGTGCAGCTGGTGCAGTC  BssHII.HuVH2: (SEQ ID NO: 2)ATCGTTTCATAAGCGCGCCAGRTCACCTTGAAGGAGTC  BssHII.HuVH3.1: (SEQ ID NO: 3)ATCGTTTCATAAGCGCGCGAGGTGCAGCTGGTGGAGTC  BssHII.HuVH3.2: (SEQ ID NO: 4)ATCGTTTCATAAGCGCGCCAGGTGCAGCTGGTGGAGTC  BssHII.HuVH3.3: (SEQ ID NO: 5)ATCGTTTCATAAGCGCGCGAAGTGCAGCTGGTGGAGTC  BssHII.HuVH4.1: (SEQ ID NO: 6)ATCGTTTCATAAGCGCGCCAGGTGCAGCTGCAGGAGTC  BssHII.HuVH4.2: (SEQ ID NO: 7)ATCGTTTCATAAGCGCGCCAGCTGCAGCTGCAGGAGTC  BssHII.HuVH5: (SEQ ID NO: 8)ATCGTTTCATAAGCGCGCGARGTGCAGCTGGTGCAGTC  BssHII.HuVH6: (SEQ ID NO: 9)ATCGTTTCATAAGCGCGCCAGGTACAGCTGCAGCAGTC  BssHII.HuVH7: (SEQ ID NO: 10)ATCGTTTCATAAGCGCGCCAGGTGCAGCTGGTGCAATC  BssHII.HuVH1.A: (SEQ ID NO: 11)ATCGTTTCATAAGCGCGCCAGGTCCAGCTTGTGCAGTC  BssHII.HuVH1.B: (SEQ ID NO: 12)ATCGTTTCATAAGCGCGCCAGGTTCAGCTGGTGCAGTC  BssHII.HuVH1.C: (SEQ ID NO: 13)ATCGTTTCATAAGCGCGCCAGGTCCAGCTGGTACAGTC  BssHII.HuVH1.D: (SEQ ID NO: 14)ATCGTTTCATAAGCGCGCCAGATGCAGCTGGTGCAGTC  BssHII.HuVH1.E: (SEQ ID NO: 15)ATCGTTTCATAAGCGCGCCAAATCCAGCTGGTGCAGTC  BssHII.HuVH1.F: (SEQ ID NO: 16)ATCGTTTCATAAGCGCGCGAGGTCCAGCTGGTGCAGTC  BssHII.HuVH3.A: (SEQ ID NO: 17)ATCGTTTCATAAGCGCGCGAGGTGCAGCTGTTGGAGTC  BssHII.HuVH3.B: (SEQ ID NO: 18)ATCGTTTCATAAGCGCGCGAGGTGCAGCTGGTGGAGAC  BssHII.HuVH4.A: (SEQ ID NO: 19)ATCGTTTCATAAGCGCGCCAGGTGCAGCTACAGCAGTG  VH Forward NheI.JH 2:(SEQ ID NO: 20) GACATTCTACGAGCTAGCTGAGGAGACAGTGACCAGGGT  NheI.JH1/4/5:(SEQ ID NO: 21) GACATTCTACGAGCTAGCTGAGGAGACGGTGACCAGGGT  NheI.JH3:(SEQ ID NO: 22) GACATTCTACGAGCTAGCTGAAGAGACGGTGACCATTGTC  NheI.JH6:(SEQ ID NO: 23) GACATTCTACGAGCTAGCTGAGGAGACGGTGACCGTGG  VK BackNheI.OL.HuVK1: (SEQ ID NO: 24)TCTCCTCACTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCGACATCCAGWTGACCCAGTC  NheI.OL.HuVK2: (SEQ ID NO: 25)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCGATGTTGTGATGACTCAGTC  NheI.OL.HuVK3: (SEQ ID NO: 26)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCGAAATTGTGWTGACRCAGTC  NheI.OL.HuVK4: (SEQ ID NO: 27)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCGATATTGTGATGACCCACAC  NheI.OL.HuVK5: (SEQ ID NO: 28)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCGAAACGACACTCACGCAGTC  NheI.OL.HuVK6: (SEQ ID NO: 29)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCGAAATTGTGCTGACTCAGTC  VK Forward NcoI.JK1−: (SEQ ID NO: 30)AGTTCATGCCATGGTTTTGATTTCCACCTTGGTCCCTT  NcoI.JK2−: (SEQ ID NO: 31)AGTTCATGCCATGGTTTTGATCTCCACCTTGGTCCC  NcoI.JK3−: (SEQ ID NO: 32)AGTTCATGCCATGGTTTTGATATCCACTTTGGTCCCAG  NcoI.JK4−: (SEQ ID NO: 33)AGTTCATGCCATGGTTTTGATCTCCAGCTTGGTCCCT  NcoI.JK5−: (SEQ ID NO: 34)AGTTCATGCCATGGTTTTAATCTCCAGTCGTGTCCCTT  VL Back NheI.OL.HuVL1.1:(SEQ ID NO: 35) TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCCAGTCTGTG CTGACTCAGCC  NheI.OL.HuVL1.2: (SEQ ID NO: 36)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCCAGTCTGTG YTGACGCAGCC  NheI.OL.HuVL1.3: (SEQ ID NO: 37)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCCAGTCTGTC GTGACGCAGCC  NheI.OL.HuVL2: (SEQ ID NO: 38)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCCARTCTGCC CTGACTCAGCC  NheI.OL.HuVL3.1: (SEQ ID NO: 39)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCTCCTATGWG CTGACTCAGCC  NheI.OL.HuVL3.2: (SEQ ID NO: 40)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCTCTTCTGAG CTGACTCAGGA  NheI.OL.HuVL4: (SEQ ID NO: 41)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCCACGTTATA CTGACTCAACC  NheI.OL.HuVL5: (SEQ ID NO: 42)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCCAGGCTGTG CTGACTCAGCC  NheI.OL.HuVL6: (SEQ ID NO: 43)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCAATTTTATG CTGACTCAGCC  NheI.OL.HuVL7/8: (SEQ ID NO: 44)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCCAGRCTGTG GTGACYCAGGA  NheI.OL.HuVL9: (SEQ ID NO: 45)TCTCCTCAGCTAGCGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCCWGCCTGTG CTGACTCAGCC  VL Forward NcoI.JL1−: (SEQ ID NO: 46)AGTTCATGCCATGGTTAGGACGGTGACCTTGGTCC NcoI.JL2/3−: (SEQ ID NO: 47)AGTTCATGCCATGGTTAGGACGGTCAGCTTGGTCC  NcoI.JL7−: (SEQ ID NO: 48)AGTTCATGCCATGGTGAGGACGGTCAGCTGGGTG

The resulting amplified cDNA products were assembled to scFv usingoverlap PCR with the following overlap primers.

(SEQ ID NO: 49) BssHII.VH.OL+: ATCGTTTCATAAGCGCGCSA (SEQ ID NO: 50)NotI.JK.OL−: AGTTCATGCCATGGTTTTGAT (SEQ ID NO: 51) NotI.JL.OL−:AGTTCATGCCATGGTKAGGAC

Purified scFv cDNA fragments (1 μg) and phagemid vector p2056BNN (2 μg)were digested with BssHII and NcoI restriction endonuclease (New EnglandBiolabs, USA). Phagemid vector p2056BNN is a modified version of pS2025e(Sidhu et al., (2004) J Mol Biol 338:299-310), engineered to containBssHII, NheI, and NcoI restriction sites. The scFv cDNA fragments werethen ligated into the p2056BNN vector (6:1 M ratio) using T4 DNA ligaseenzyme (New England Biolabs). The resulting cDNA/phage ligation productswere purified using a PCR purification kit (Qiagen, USA) and transformedinto electro-competent SS320 E. coli cells. The size of the phagelibrary was estimated by plating 10 μl of 1:10 diluted library cultureonto LB/Carbenicillin plates. The library culture was then furtheramplified and propagated in a total volume of 60 ml 2YT medium, andphage-scFv expression was induced by co-infection with M13KO7 helperphage. Kanamycin was later added to the library culture, and incubatedwith shaking for 30 hours at 30° C. The library culture was thencentrifuged to pellet the cells. The phage-scFv-containing supernatantwas precipitated with 5×PEG/2.5 M NaCl and resuspended in PBS.

Phage Library Sorting and Screening to Identify Anti-HemagglutininAntibodies

Influenza A virus hemagglutinin H1 and H3 proteins (produced asdescribed below in Example 2) were used as antigens for phage librarysorting. Hemagglutinin H1 and H3 antigens were coated onto ahigh-binding 96-well maxisorp plate. The plates and phage libraries werepre-blocked with phage blocking buffer (phosphate-buffered saline (PBS),1% (w/v) bovine serum albumin (BSA), and 0.05% (v/v) tween-20 (PBS-T))and incubated for 2 hours at room temperature. The blocked phage library(100 μl) was added to the hemagglutinin-coated wells and incubated for 3hours. The unbound phage were washed off the plates using 0.05%PBS-Tween, and bound phage were eluted with 100 μL 50 mM HCl and 500 mMNaCl for 30 minutes followed by neutralization with 100 μL of 1 M Trisbase (pH 7.5). Recovered phage were amplified in E. coli XL-1 Bluecells. The resulting phage were precipitated and subjected another roundof panning/selection against the hemagglutinin proteins. Duringsubsequent panning/selection rounds, antibody phages were incubated withsame or different hemagglutinin antigens. The stringency of platewashing was gradually increased from washing 15× to washing 40×.

After 2-3 rounds of panning and selection, significant enrichment ofhemagglutinin-specific phage was observed. 96 phage clones were pickedfrom the library sorting to determine whether they specifically bound tohemagglutinin H1 and/or H3. The variable regions of the phage clonesdisplaying specific binding to the hemagglutinin proteins were sequencedto identify phage clones containing unique immunoglobulin nucleic acidsequences. Unique phage antibodies that bound hemagglutinin H1 and/or H3with at least 5× above background were further characterized.Phage-derived clones of interest were reformatted into IgGs by cloningV_(L) and V_(H) regions of individual clones into the LPG3 and LPG4expression vectors, respectively, transiently expressed in mammalian 293cells, and purified using a protein A column. Two antibodies (mAb9 andmAb23) were identified for further analysis. (See Example 5 below.)

Example 2 Plasmablast Enrichment and Expansion

To discover and identify rare antibodies against influenza A virushemagglutinin, the following plasmablast enrichment and expansiontechnique was developed. (See co-pending patent application U.S. patentapplication Ser. No. 61/725,764, which is incorporated by referenceherein in its entirety.)

Leukopacs from normal human donors that received the seasonal influenzaFluvirin® vaccine (Novartis Lot #111796P1) 7 days prior to their blooddonation were obtained from Blood Centers of the Pacific (San Francisco,Calif.). Peripheral blood mononuclear cells (PBMCs) were isolated fromthe leukopacs using standard methodologies. Six- to eight-week oldfemale SCID/beige mice were purchased from Charles River Laboratories(Hollister, Calif.) and housed and maintained at Genentech in accordancewith American Association of Laboratory Animal Care guidelines. Allexperimental studies were conducted under the approval of theInstitutional Animal Care and Use Committees of Genentech Lab AnimalResearch in an AAALACi-accredited facility in accordance with the Guidefor the Care and Use of Laboratory Animals and applicable laws andregulations. Leukopac or blood from healthy human donors was obtainedafter written informed consent was provided and ethical approval grantedfrom the Western Institutional Review Board.

In vivo antigen-driven plasmablast enrichment and expansion wasperformed using intraspenic transplantation of PBMCs as follows.Isolated PBMCs were resuspended with hemagglutinin antigens (0.1-2 μgfor each one million B cells) and incubated for 30 minutes at 37° C.(PBMC/antigen pre-mix). Following this incubation, the PBMCs were washedto remove unbound antigens. To enrich for plasmablasts that producedcross-reactive hemagglutinin antibodies, the hemagglutinin antigenvariants used for PBMC/antigen pre-mix and single cell sorting werespecifically chosen to differ from the hemagglutinin antigen variantscontained within the influenza Fluvirin® vaccine. Hemagglutinin antigensused in this study, therefore, included H1 hemagglutinin from influenzaA virus isolate A/NWS/1933 (a Group1 influenza A virus hemagglutinin),H3 hemagglutinin from influenza A virus isolate A/Hong Kong/8/1968 (aGroup2 influenza A virus hemagglutinin), and H7 hemagglutinin frominfluenza A virus isolate A/Netherlands/219/2003 (a Group2 influenza Avirus hemagglutinin) The hemagglutinin antigens were produced atGenentech using standard molecular biology techniques.

6-8 week old female SCID/beige mice (Charles River Laboratories,Hollister, Calif.) were sub-lethally irradiated with 350 rads using aCesium-137 source. Polymyxin B (110 mg/L) and neomycin (1.1 g/L) wereadded to the drinking water for 7 days following irradiation. Four hoursafter irradiation, the left flank of each mouse was shaved and preppedwith Betadine® (Purdue Pharma, Stamford, Conn.) and 70% alcohol.Surgical procedures were performed under anesthesia using asepticsurgical procedures. A 1-cm skin incision was made just below the costalborder of each mouse, followed by an incision of the abdominal wall andthe peritoneum. The spleen of each mouse was carefully exposed andinjected with 50×10⁶ human PBMCs resuspended in 30 μL PBS. The incisionswere closed in the muscular layer and in the skin using 5-O Vicryl®sutures (Ethicon, Somerville, N.J.) and surgical staples, respectively.For antigen-specific cell sorting experiments, mice were sacrificed at 8days post-transplantation, and their spleens harvested. Single cellsuspensions of spleen cells obtained from the mice were stained with acocktail of anti-human monoclonal antibodies CD38 PECy7 (BD Biosciences,San Jose, Calif.) and IgG Dylight (Jackson ImmunoResearch Laboratories,Inc., West Grove, Pa.) which define human IgG+ plasmablasts asCD38^(high)/IgG+ expression. To identify hemagglutinin cross-reactiveplasmablasts within the suspension of isolated spleen cells, the cellswere stained with hemagglutinin H1 from influenza virus A isolateA/NWS/1933 and hemagglutinin H3 from influenza virus A isolate A/HongKong/8/1968, which were previously conjugated with FITC or PE,respectively, using Lightning-Link® labeling kits (Innova Biosciences,Cambridge, UK).

FIG. 1A shows representative FACS data analysis ofanti-hemagglutinin-positive plasmablasts from day 7 post-vaccinatedPBMCs prior to SCID/beige mice enrichment (i.e., prior to PBMC/antigenpre-mix). FIG. 1B shows representative FACS data analysis ofhemagglutinin-positive plasmablasts from day 8 post-transplant afterSCID/beige mice enrichment, comparing no pre-mix and antigen pre-mix inthe upper and lower panels, respectively. As shown in FIGS. 1A and 1B,PBMC/antigen pre-mix prior to intrasplenic injection resulted in higherfrequency of H3⁺/H1⁺ anti-hemagglutinin plasmablasts.

Table 2 below shows a comparison of anti-H1⁺/anti-H3⁺ plasmablastfrequencies before and after SCID enrichment as described herein. Asshown in Table 2, the frequency of anti-H1⁺/anti-H3⁺ plasmablasts wasgreatly increased using the SCID/beige mouse enrichment methods of thepresent invention compared to that observed without SCID/beige mouseenrichment.

TABLE 2 Anti-H1⁺/Anti-H3⁺ Plasmablast Condition Frequency (%) VaccinatedPBMC 0.00028 ± 0.00008 SCID + Antigen Premix 0.011 ± 0.007

Samples were then analyzed in the presence of propidium iodide dead cellexclusion on Aria high-speed cell sorter (BD Biosciences, San Jose,Calif.) and anti-hemagglutinin-specific plasmablasts were sorted in asingle cell manner into 96-well tissue culture plates containing 50 μlRPMI cell cutlute media supplemented with 5% Low IgG fetal bovine serum.(Gibco, Grand Island, N.Y.). Five million live cells were recorded forall analysis profiles. Profiles were analyzed by Flowjo version 9.4.11software.

FIG. 2 shows analysis of splenocytes obtained from day-8 post-transplantfrom individual SCID/beige mice showing stochastic response, comparingno pre-mix (circles) and antigen-pre-mix (squares). Data is presented aspercent anti-H1⁺/CD38^(high) plasmablasts. The rectangle indicates micethat presented anti-H1⁺ plasmablasts.

These results showed that broad hemagglutinin cross-reactiveplasmablasts were detected if influenza virus A Group1 (e.g.,hemagglutinin H1) and Group2 (e.g., hemagglutinin H3, hemagglutinin H7)hemagglutinin antigens were incubated with PBMCs prior to intrasplenictransplant. These results further indicated that in vitro stimulation ofhemagglutinin antigen-primed PBMCs from influenza-vaccinated donorspromoted hemagglutinin antigen-specific enrichment of plasmablastswithin the SCID/beige mouse recipients.

Example 3 IgG Cloning from Single Plasmablasts

Hemagglutinin H1 and H3 cross-reactive human plasmablasts (describedabove) were single-cell sorted, resulting in approximately 950plasmablasts. Single plasmablasts were sorted directly into U-bottom96-well micro-well plates containing 50 μl RPMI containing 5% Low IgGfetal bovine serum. The plates were centrifuged for 5 minutes at 600×g(Beckman Coulter, Brea, Calif.) and the media was carefully removed byaspiration. The cells were re-suspended and washed twice in 90 μl of PBSfollowing the same procedure.

To generate cDNA encoding the variable heavy chains and light chains,each cell was re-suspended in 6 μl of Reverse Transcriptase (RT)reaction mixture containing 2 units RNaseout (Invitrogen, Grand Island,N.Y.), 0.5 mM 4dNTP (Perkin Elmer, Waltham, Mass.), 1.5 mM MgCl₂, 37.5mM KCl, 10 mM DTT (dithiothreitol), 0.25% Nonidet P40 (US Biological,Marblehead, Mass.), 0.1 mg/ml bovine serum albumin (Sigma-Aldrich), 25mM Tris pH 8.3, 0.25 pmol of IgG₁₋₄ constant, kappa chain constant, andlambda chain constant region specific oligonucleotides (shown below) and40 U Superscript III (Invitrogen, Grand Island, N.Y.).

(SEQ ID NO: 52) IgG₁₋₄ constant: GAAGTAGTCCTTGACCAGGCAG (SEQ ID NO: 53)Kappa constant: CTCAGCGTCAGGGTGYTGCTGAG (SEQ ID NO: 54) Lambda constant:GGGTKTGGTSGTCTCCAC

The reaction was incubated for 3×30-minute intervals at 45° C., 50° C.,and 55° C. each. Following the incubation, the reaction mixture wasdiluted to 15 μl with TE buffer (10 mm Tris HCl, 1 mM EDTA). Initialpolymerase chain reactions (PCR) were performed to amplify IgG heavychains, kappa chains, and lambda chains using 2 μl of the diluted RTcocktail from above and Advantage-GC 2 Polymerase Mix (Clontech,Mountain View, Calif.), following protocols provided by themanufacturers. The PCR amplifications were performed using degenerateoligonucleotides based on variable heavy chain and light chain germlineand constant region sequences shown below.

(SEQ ID NO: 55) IGVH1a CAGGTGCAGCTGGTGCAGTCTGGGGC (SEQ ID NO: 56) IGVH1bCAGGTCCAGCTGGTGCAGTCTGGGGC (SEQ ID NO: 57) IGVH2CAGGTCACCTTGAAGGAGTCTGGTCC (SEQ ID NO: 58) IGVH3GAGGTGCAGCTGGTGGAGTCTGGGGG (SEQ ID NO: 59) IGVH4CAGGTGCAGCTGCAGGAGTCGGGCCC (SEQ ID NO: 60) IGVH5 GAGGTGCAGCTGGTGCAGTCTGG(SEQ ID NO: 61) IGVH6 CAGGTACAGCTGCAGCAGTCAGGTCC (SEQ ID NO: 62) IGVH7CAGGTGCAGCTGGTGCAATCTGG (SEQ ID NO: 63) IGKV1 GHCATCCRGWTGACCCAGTCTC(SEQ ID NO: 64) IGKV2 GATRTTGTGATGACYCAGWCTC (SEQ ID NO: 65) IGKV3GAAATWGTRWTGACRCAGTCTC (SEQ ID NO: 66) IGKV4 GACATCGTGATGACCCAGTCTCC(SEQ ID NO: 67) IGKV5 GAAACGACACTCACGCAGTCTC (SEQ ID NO: 68) IGKV6GAWRTTGTGMTGACWCAGTCTC (SEQ ID NO: 69) IGLV1 CAGTCTGTGYTGACKCAGCCRCCCTC(SEQ ID NO: 70) IGLV2 CAGTCTGCCCTGACTCAGCCT (SEQ ID NO: 71) IGLV3TCCTATGAGCTGACWCAGSHVCCCKC (SEQ ID NO: 72) IGLV4CAGCCTGTGCTGACTCARTCVCCCTC (SEQ ID NO: 73) IGLV5CAGCCTGTGCTGACTCAGCCAACTTC (SEQ ID NO: 74) IGLV6AATTTTATGCTGACTCAGCCCCAC (SEQ ID NO: 75) IGLV7 CAGGCTGTGGTGACTCAGGAGCCC(SEQ ID NO: 76) IGLV8 CAGACTGTGGTGACCCAGGAGCC (SEQ ID NO: 77) IGLV9CAGCCTGTGCTGACTCAGCCACC (SEQ ID NO: 78) HC301.5 constantGCAGCCCAGGGCSGCTGTGC (SEQ ID NO: 79) Kappa102constantGCACACAACAGAGGCAGTTCCAG (SEQ ID NO: 80) Lambda202constantCTTGRAGCTCCTCAGAGGAG

Heavy chain and light chain PCR amplification reactions were eachdivided into two reactions as follows: heavy chain families VH.1,2,3(primers IGVH1a, IGVH1b, IGVH2, IGVH3) and VH.4,5,6,7 (primers IGVH4,IGVH5, IGVH6, and IGVH7); kappa chain families VK.1,2,3 (primers IGKV1,IGKV2, and IGKV3) and VK.4,5,6 (primers IGVK4, IGVK5, and IGVK6); andlambda chain families VL.1,2,3,4,5 (IGLV1, IGLV2, IGLV3, IGLV4, andIGLV5) and VL.6,7,8,9 (primers IGLV6, IGLV7, IGLV8, and IGLV9). Atouchdown PCR amplification protocol was used for temperature cycling.

Following the reaction, PCR amplification products were treated withExonuclease1 (Exo) and Shrimp Alkaline Phosphatase (SAP) to removeexcess nucleotides and primers from each of the PCR amplificationreactions (U.S. Biologicals, Marblehead, Mass.). Initial PCRamplification products were directly sequenced to determine the variablesequences of both the heavy chains and light chains using Sangersequencing. Second nested PCR amplifications were performed usinggermline-matched heavy chain and light chain variable oligonucleotidesin order to insert a mammalian signal and constant region cloningsequences using the following oligonucleotide primers.

sVH1a: (SEQ ID NO: 81)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACT GGAGTACATTCACAGGsVH2:  (SEQ ID NO: 82)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGATCACCT sVH3vv: (SEQ ID NO: 83)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACT GGAGTACATTCACAGsVH3g1: (SEQ ID NO: 84)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACT GGAGTACATTCAGAGGsVH4: (SEQ ID NO: 85) CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGGTGCAGCTGCAGG sVH5: (SEQ ID NO: 86)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACT GGAGTACATTCAGAGGTGCAsVH6: (SEQ ID NO: 87) CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGGTACAGC sVH7: (SEQ ID NO: 88)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACT GGAGTACATTCACAGGTGCAsVK1: (SEQ ID NO: 89) CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGACATCCAGATGACCCAGTCTCCATCCTCCCTG sVK2: (SEQ ID NO: 90)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGATATTGTGATGACTCAGTCTCACTCTCCCTGC sVK3: (SEQ ID NO: 91)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTT G sVK4:(SEQ ID NO: 92) CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGT G sVK5:(SEQ ID NO: 93) CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGAAACGACACTCACGCAGTCTCCAGC sVK6: (SEQ ID NO: 94)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGAAATTGTGCTGACTCAGTCTCCAGACTTTCG sVL1: (SEQ ID NO: 95)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGTCTGTGYTGACKCAGCCRCCCTC sVL2: (SEQ ID NO: 96)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGTCTGCCCTGACTCAGCCT sVL3: (SEQ ID NO: 97)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCATCCTATGAGCTGACWCAGSHVCCCKC sVL4: (SEQ ID NO: 98)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGCCTGTGCTGACTCARTCVCCCTC sVL5: (SEQ ID NO: 99)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGCCTGTGCTGACTCAGCCAACTTC sVL6: (SEQ ID NO: 100)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAAATTTTATGCTGACTCAGCCCCAC sVL7: (SEQ ID NO: 101)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGGCTGTGGTGACTCAGGAGCCC sVL8: (SEQ ID NO: 102)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGACTGTGGTGACCCAGGAGCC wVL9: (SEQ ID NO: 103)CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGCCTGTGCTGACTCAGCCACC Heavy constant: (SEQ ID NO: 104)GCCAGGGGGAAGACCGATG Kappa constant: (SEQ ID NO: 105)CTGGGATAGAAGTTATTCAGCAGGCACACAACAGAAGCAGTTCCAGATTT CAACTGCTCLambda constant: (SEQ ID NO: 80) CTTGRAGCTCCTCAGAGGAG

PCR amplification reactions were set up using PrimeStar HS DNAPolymerase with GC (Takara Bio, Shiga, Japan) according to themanufacturer's recommendation. Following the PCR amplificationreactions, the amplification products were treated with Exo/SAP asdescribed above. Heavy variable chain and light variable chain encodingPCR amplification products were inserted into a mammalian expressionvector using restriction endonuclease free procedures. 20 μl of the PCRamplification products were annealed onto single stranded DNA humantemplates for IgG₁, kappa, and lambda chain using the Kunkel mutagenesisprotocol. (See Kunkel (1985) PNAS 82:488-492.) Correctly insertedconstructs were confirmed by DNA sequencing. Plasmids containing nucleicacids encoding heavy chains and light chains were co-transfected into293T human embryonic kidney cells using Fugene transfection reagent(Roche Diagnostic, Indianapolis, Ind.) for transient expression, andanalyzed for expression and binding as described below in Example 4.

Example 4 Hemagglutinin ELISA Screening Assay

The ability of each monoclonal anti-hemagglutinin antibody obtained asdescribed above to bind various hemagglutinin subtypes was examined byELISA as follows. Various hemagglutinin-expressing plasmids weretransfected into 293T cells as described above. These includedhemagglutinin H1 from H1N1/South Carolina/1918, hemagglutinin H3 fromH3N2/Perth/2009, hemagglutinin H5 from H5N1/Viet/2004, and hemagglutininH7 from H7N7/Netherlands/2003 influenza A viruses. After two days, cellswere lysed in 50 mM Tris, pH 8, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100plus protease inhibitor cocktail (Roche). Nuclei were cleared bycentrifugation and the resulting lysates were stored at −80° C.

For ELISA screening, 384-well plates (Nunc MaxiSorp) were coated with 5μg/ml Galanthus nivalis lectin (Sigma) in PBS. The plates were washedand then coated with dilutions of the cell lysates containing variousexpressed hemagglutinins. The plates were washed and incubated withvarious dilutions of the anti-hemagglutinin antibodies and subsequentlywith a goat-anti-human-HRP secondary antibody (Jackson). Plates werewashed and processed for TMB (3,3′,5,5′-tetramethylbenzidine) substratedetection.

Approximately 950 plasmablasts were obtained from single-cell sortingdescribed above in Example 2. Of this, 840 monoclonal antibodies weretransiently expressed in 293T cells and screened by ELISA for binding tohemagglutinin subtypes H1, H3, H5, and H7, resulting in 82 monoclonalantibodies that bound influenza A virus Group1 or Group2 hemagglutinin,and 20 monoclonal antibodies that bound both influenza A virus Group1and Group2 hemagglutinins

Example 5 In Vitro Influenza a Virus Neutralization

The ability of the anti-hemagglutinin antibodies of the presentinvention to elicit broad hemagglutinin subtype binding andneutralization of a panel of influenza A Group1 and Group2 virusisolates in vitro was examined as follows.

MDCK cells were grown in DMEM media supplemented with 10% FBS as asingle 25% confluent monolayer in 96-well black with clear bottomimaging plates (Costar 3904). Each influenza A virus subtype/strain wasdiluted in influenza media (DMEM+0.2% BSA, 2 μg/ml TPCK treated Trypsin)to an MOI of 1 and incubated for 1 hour at 37° C. with varyingconcentrations (ranging from 0.02 nM to 1,600 nM) of each antibody. Eachantibody/influenza virus mixture was allowed to infect MDCK cells for 16hours at 37° C. in a 5% CO₂ incubator prior to fixation of the cellswith cold 100% ethanol. The fixed cells were then stained with Hoechst33342 (Invitrogen, Cat#H3570) to visualize cell nuclei and determinetotal cell number. The cells were also stained with a broadly reactivemonoclonal antibody (Millipore Cat# MAB8258) specific for influenza Avirus nucleoprotein in order to determine the number of infected cells.

Cells were imaged using the Image Express Micro (Molecular Devices) anddata images were analyzed using MetaXpress 3.1 software. The percentageof infected cells was determined and plotted on the Y-axis versus theLog 10 antibody concentration on the X-axis. All neutralization assayswere completed in triplicate. Data were fit using a nonlinear regressiondose-response curve and are presented in FIG. 3 as IC₅₀ values in nMwith 95% confidence intervals (95% CI). The hemagglutinin (HA) subtypeof each influenza A virus strain is provided in the table shown in FIG.3.

In vitro neutralization dose-response curves were generated usingvarious concentrations of the monoclonal antibodies described hereinagainst a broad panel of influenza A Group1 and Group2 virus strains.FIGS. 4A and 4B show neutralization curves of mAb 39.29 NWPP (“NWPP”disclosed as SEQ ID NO: 177) against a panel of influenza A Group1 andGroup2 virus strains, respectively. As shown in FIGS. 4A and 4B, mAb39.29 NWPP (“NWPP” disclosed as SEQ ID NO: 177) was effective at invitro neutralization of all influenza A virus strains tested. (See alsoFIG. 3.) Additionally, FIGS. 5A and 5B show neutralization curves of mAb81.39 SVSH-NYP (“SVSH” disclosed as SEQ ID NO: 171) against a panel ofinfluenza A Group1 and Group2 virus strains, respectively. As shown inFIGS. 5A and 5B, mAb 81.39 SVSH-NYP (“SVSH” disclosed as SEQ ID NO: 171)was effective at the in vitro neutralization of all influenza A virusstrains tested. (See also FIG. 3.)

Four anti-hemagglutinin antibodies of the present invention(specifically mAb 39.18 B11, mAb 36.89, mAb9.01F3, and mAb23.06C2) wereeffective in vitro at neutralization of either Group1 or Group2influenza A virus strains, but not both. Specifically, mAb 39.18 B11 waseffective at in vitro neutralization of the entire Group1 influenza Avirus panel examined, but was not able to neutralize Group2 influenza Avirus strains. (See FIG. 6 and FIG. 3.) Conversely, mAb 36.89,mAb9.01F3, and mAb23.06C2 were able to neutralize the entire Group2influenza A virus panel examined, but were not able to neutralize anyGroup1 influenza A virus isolate tested. (See FIGS. 7, 8, and 9, showingin vitro neutralization curves for mAb 36.89, mAb9.01F3, and mAb23.06C2,respectively; also see FIG. 3.)

Taken together, these results showed that monoclonal antibodies of thepresent invention were able to neutralize in a dose-dependent mannervarious influenza A virus isolates/strains in vitro. Additionally, theseresults showed that the plasmablast enrichment methodology describedherein resulted in the identification of monoclonal antibodies capableof neutralizing both Group1 and Group2 influenza A virus strains fromonly 950 isolated plasmablasts.

In vitro neutralization studies were also performed using a pseudotypevirus engineered to express hemagglutinin H5 to test the efficacy of anantibody of the present invention at neutralizing H5N1 influenza Avirus. In particular, an HIV psueudotype virus bearing the H5hemagglutinin surface protein was tested for neutralization with mAb39.29 NCv1 on 293T cells as follows. The H5 pseudotype virus wasproduced by co-transfection of 293T cells with three plasmids: Δ8.9,FCMV-GFP, and a plasmid expressing hemagglutinin H5 from influenza Avirus isolate H5N1/Vietnam/1203/2004. Virus was purified byultra-centrifugation through 20% sucrose.

For infection, pseudotype virus was incubated with various amounts ofmAb 39.29 NCv1 before adding to target 293T cells cultured in 96-wellplates. After two days, the number of infected cells was determined bycounting GFP positive cells. Infection was normalized to the number ofinfected cells at the lowest antibody concentration used. The resultsare presented in FIG. 10. As shown in FIG. 10, mAb 39.29 NCv1 displayeda dose-dependent in vitro neutralization against the pseudotype virusexpressing hemagglutinin H5 surface protein. These data suggested thatantibodies of the present invention would be effective at treatment andprevention of H5N1 influenza A virus strains.

An equine influenza virus was also tested for the ability of antibodiesof the present invention to exhibit in vitro neutralization activity asfollows. H7N7 A/Equine/1/Prague/56 influenza A virus was passed on MDCKcells until it achieved a high degree of infectivity. The resulting H7N7A/Equine/1/Prague/56 influenza A virus was used in neutralization assays(using methods as described above for mAb 39.29 NCv1) on MDCK cells. Theresults of these experiments are presented in FIG. 11. As shown in FIG.11, mAb 39.29 NWPP (“NWPP” disclosed as SEQ ID NO: 177) displayed adose-dependent in vitro neutralization against the H7N7A/Equine/1/Prague/56 influenza virus expressing hemagglutinin H7 surfaceprotein.

Taken together, these results showed that anti-hemagglutinin antibodiesof the present invention exhibited dose-dependent neutralizationactivity against a variety of influenza A virus strains. Specifically,two anti-hemagglutinin antibodies (mAb 39.29 NWPP (“NWPP” disclosed asSEQ ID NO: 177) and mAb 81.39 SVSH-NYP (“SVSH” disclosed as SEQ ID NO:171)) were effective at neutralizing all influenza A virus strainsexamined, including neutralization of both Group1 influenza A virusstrains (A/CA/7/2009, A/Brisbane/59/2007, A/Solomon/3/2006, A/NewCaledonia/20/1999, A/PR/8/1934, and A/Japan/305/1957) and Group2influenza A virus strains (A/Victoria/361/2011, A/Perth/16/2009,A/Brisbane/10/2007, A/Wisconsin/67/2005, A/Victoria/3/1975, A/PortChalmers/1/1973, A/HK/8/1968, and A/Aichi/2/1968).

Additionally, these results showed that anti-hemagglutinin antibodies ofthe present invention (e.g., mAb 39.29 NWPP (“NWPP” disclosed as SEQ IDNO: 177) (FIGS. 4A and 4B) and mAb 81.39 SVSH-NYP (“SVSH” disclosed asSEQ ID NO: 171) (FIGS. 5A and 5B)) were effective at neutralization of avariety of different seasonal H1N1 influenza A virus strains, H3N2influenza A virus strains, a H2N2 influenza A virus strain, and theinfluenza A virus strain associated with the 1957 Japan pandemic(A/Japan/305/1957). These results indicated that antibodies of thepresent invention are effective in the treatment and prevention ofseasonal influenza A virus infection and influenza A virus strainsassociated with influenza pandemics.

Example 6 In Vivo Efficacy of mAb 39.29 NWPP (“NWPP” Disclosed as SEQ IDNO: 177) in Mice

The in vivo efficacy of mAb 39.29 NWPP (“NWPP” disclosed as SEQ ID NO:177) to influenza A virus infection in mice was performed as follows.DBA/2J mice (Jackson Lab, Bar Harbor, Me.) were infected intranasallywith 50 μl of various influenza A virus strains diluted in influenzamedia (DMEM, 0.2% BSA, 2 μg/mL TPCK-treated trypsin) at the minimumLD₁₀₀ dose. Four different influenza A virus strains exhibiting a rangeof in vitro IC₅₀ values were used in this series of experiments,including: H1N1 A/PR/8/1934 (Genentech; IC₅₀ 2.0 nM), used at 40 PFU permouse; H3N2 A/Hong Kong/1/1968 (ViraPur, San Diego, Calif.; IC₅₀ 45.1nM), used at 3 PFU per mouse; H3N2 A/Port Chalmers/1/1973 (ViraPur, SanDiego, Calif.; IC₅₀ 2.2 nM), used at 1.5×10⁴ PFU per mouse; and H3N2A/Aichi/2/1968 (ViraPur, San Diego, Calif.; IC₅₀ 35 nM), used at 2×10²PFU per mouse. Influenza virus infection was allowed to progress for 72hours prior to the intravenous administration of mAb 39.29 NWPP (“NWPP”disclosed as SEQ ID NO: 177).

After 72 hours post influenza virus A infection, various amounts of mAb39.29 NWPP (“NWPP” disclosed as SEQ ID NO: 177) were administeredintravenously to the mice at a dose of 900 μg/mouse (approximately 45mg/kg), 300 μg/mouse (approximately 15 mg/kg), and 100 μg/mouse(approximately 5 mg/kg) in 200 μl PBS. Control treated animals wereadministered mAb gD5237 (a monoclonal antibody specific for glycoproteinD of herpes simplex virus (HSV)) at the highest tested equivalent doseof mAb 39.29 NWPP (“NWPP” disclosed as SEQ ID NO: 177) (i.e.,approximately 45 mg/kg). Mice were monitored daily for body conditioningand survival, and also weighed daily, until 21 days after infection. AllmAb39.29 NWPP (“NWPP” disclosed as SEQ ID NO: 177) doses vs. control inall four influenza A virus strain infections gave a Log-rank test ofP<0.01.

FIGS. 12A, 12B, 12C, and 12D show percent survival (over time, in days)of mice administered various amounts of mAb 39.29 NWPP (“NWPP” disclosedas SEQ ID NO: 177) 72 hours after infection with influenza A virusA/PR/8/1934, A/Port Chalmers/1/1973, A/Hong Kong/1/1968, andA/Aichi/2/1968, respectively. As shown in FIGS. 12A, 12B, 12C, and 12D,100% mortality was observed by day 14 in infected mice administeredcontrol antibody. However, infected mice administered monoclonalantibody of the present invention showed increased survival. Inparticular, 100% survival was observed in mice infected with influenzavirus A/Port Chalmers/1/1973 or influenza virus A/Aichi/2/1968 at alldoses of mAb 39.29 NWPP (“NWPP” disclosed as SEQ ID NO: 177) tested.(See FIGS. 12B and 12D.)

These results showed that monoclonal antibodies of the present inventionare effective at treating various influenza A virus infections.Additionally, these data showed that monoclonal antibodies of thepresent invention were effective at treating influenza A virus infectionwhen administered up to at least 72 hours post influenza A virusinfection.

Example 7 In Vivo Efficacy of mAb 39.29 NCv1 in Mice

To test the in vivo efficacy of mAb 39.29 NCv1 in mice, the antibody wasadministered i.v. to mice infected with four different influenza A virusisolates that exhibited a range of in vitro IC₅₀ values. DBA/2J mice(Jackson Lab, Bar Harbor, Me.) were infected intranasally with 50 μl ofdifferent influenza A virus strains diluted into influenza media (DMEM,0.2% BSA, 2 ug/mL TPCK treated trypsin) at the minimum LD100 dose.

In one set of experiments, influenza A virus isolate H1N1 A/PR/8/1934was used at 40 PFU per mouse. At 72 hours post infection,anti-hemagglutinin mAb 39.29 NCv1 was administered intravenously atapproximately 15 mg/kg, approximately 5 mg/kg, approximately 1.7 mg/kg,or approximately 0.56 mg/kg in 200 μl PBS intravenously. Control treatedanimals were given mAb gD5237, which is specific for glycoprotein D ofHSV at the highest tested equivalent dose of mAb 39.29 NCv1. Mice weremonitored for body conditioning and survival, and weighed until 21 daysafter infection.

For the H1N1 A/PR/8/1934 infected mice, a single i.v. dose of mAb 39.29NCv1 at 15 mg/kg per mouse was efficacious compared to that observedwith control IgG antibody. (See FIG. 13.) Specifically, 100% mortalitywas observed in the control treatment group by day 12, while a singledose of 15 mg/kg of mAb 39.29 NCv1 saved 87.5% of the infected mice. Athreefold lower dose of 100 μg per mouse (approximately 5 mg/kg) of mAb39.29 NCv1 exhibited some efficacy, being able to protect 25% of animalsfrom the lethal challenge, while doses of approximately 1.7 mg/kg orapproximately 0.56 mg/kg showed minimal efficacy beyond that observed inthe control treatment group. (See FIG. 13.)

In another set of experiments, in vivo efficacy of mAb 39.29 NCv1 wasfurther examined against mouse-adapted H3N2 Hong Kong influenza A virusstrain (H3N2 A/Hong Kong/1/1968), which has a tenfold higher in vitroIC₅₀ than A/PR8/1934. As observed in previous experiments describedabove, mice treated with control antibody following influenza A virusinfection showed 100% mortality by day 12. (See FIG. 14.) However, asingle dose of mAb 39.29 NCv1 at approximately 45 mg/kg or approximately15 mg/kg was able to protect 87.5% and 75% of the mice, respectively.The minimum efficacious dose of 15 mg/kg in vivo of mAb 39.29 NCv1 inboth the A/PR8/1934 and the A/Hong Kong/1/1968 influenza A virusinfection models is very similar despite the observed contrast in mAb39.29 NCv1 in vitro IC₅₀ values between these two strains. (See FIGS. 3and 14.)

To further explore the in vivo efficacy of mAb 39.29 NCv1, a dosetitration of mAb 39.29 NCv1 was tested against two additional influenzaA virus strains, Port Chalmers (H3N2 A/Port Chalmers/1/1973) and Aichi(H3N2 A/Aichi/2/1968). mAb 39.29 NCv1 has an in vitro IC₅₀ against PortChalmers of 2.9 nM, which is very similar to that of A/PR8/1934, whileAichi has an in vitro IC₅₀ of 35.0 nM, a value closer to that of A/HongKong/1/1968. As shown in FIG. 15 and FIG. 16, 100% mortality wasobserved in the control treated animals by day 12 and day 10 for thePort Chalmers and Aichi models, respectively. Monoclonal antibody 39.29NCv1 exhibited very efficacious against both influenza A virus strainsat all tested doses (e.g., 45 mg/kg, 15 mg/kg, 5 mg/kg, and 1.7 mg/kg).

These data indicated, in part, that little correlation existed betweenthe in vitro IC₅₀ of mAb 39.29 NCv1 and the in vivo minimum efficaciousdose. None-the-less, a single dose of 15 mg/kg administered i.v. 72hours post infection was efficacious in all four influenza A virus mousemodels despite the range of in vitro IC₅₀ values for these influenza Avirus strains.

Example 8 In Vivo Efficacy of mAb 39.29 and Oseltamivir in SevereInfluenza a Virus Infection in Mice

To compare the efficacy of anti-hemagglutinin antibodies of the presentinvention to that of oseltamivir phosphate (Tamiflu®) in mice, thefollowing studies were performed. Balb/c mice (Charles RiverLaboratories, Hollister, Calif.) at 6-weeks old were infectedintranasally with 50 μl H1N1 A/PR/8/1934 at 100× the lethal dose (5×10⁴PFU/mouse). At 48 hours post infection, anti-hemagglutinin antibody39.29 (a 50:50 mixture of mAb 39.29 D8C2 and mAb 39.29 NWPP (“NWPP”disclosed as SEQ ID NO: 177)) was administered as a single dose ofapproximately 15 mg/kg or control IgG in 200 μl PBS intravenously. Inthese experiments, an oseltamivir dosing regimen consisting of 2 mgdosed twice daily (BID) for five days was compared with a single 300 μgi.v. dose (˜15 mg/kg) of mAb 39.29 NWPP (“NWPP” disclosed as SEQ ID NO:177). A Log-rank test of mAb 39.29 NWPP (“NWPP” disclosed as SEQ ID NO:177) or oseltamivir vs. control gave p<0.01 and a maximum likelihoodtest of mAb 39.29 NWPP (“NWPP” disclosed as SEQ ID NO: 177) vs.oseltamivir gave p<0.05. (Oseltamivir (i.e., Tamiflu®) was obtained fromToronto Research Chemicals, Cat. No. 0701000.)

As shown in FIG. 17, 100% mortality was observed by day 9 in control-IgG(mAb gD5237) treated animals. BID treatment of oseltamivir for 5 daysonly protected 37.5% of mice from lethality. However, a single 15 mg/kgdose of mAb 39.29 NWPP (“NWPP” disclosed as SEQ ID NO: 177) mixtureprotected 87.5% of the infected animals from the lethal influenza Avirus challenge. (See FIG. 17.) The fully efficacious 15 mg/kg dose ofmAb 39.29 NWPP (“NWPP” disclosed as SEQ ID NO: 177) mixture performedbetter than oseltamivir in mice severely infected with influenza Avirus.

These results showed that a single dose of a monoclonal antibody of thepresent invention was more effective at treating influenza A virusinfection than a 5-day treatment with oseltamivir.

Example 9 In Vivo Efficacy of mAb 39.29 NWPP (“NWPP” Disclosed as SEQ IDNO: 177) in Mice with and without Co-Administration of Oseltamivir

Administration of oseltamivir is effective at reducing human influenza Avirus infection if given within 48 hours after symptom onset.Unfortunately, oseltamivir shows minimal efficacy in patients who havebeen symptomatic for more than 48 hours. Therefore, the followingexperiments were performed to test if co-administration of a monoclonalantibody of the present invention and oseltamivir showed improvedefficacy over either treatment alone. These experiments were performedusing the severe mouse influenza infection model described above inExample 8. Briefly, female Balb/C mice (Charles River Laboratories) wereinfected with 100× the lethal dose (5×10⁴ pfu) of A/PR/8/1934 72-hoursprior to i.v. administration of a single dose of 100 μg mAb 39.29 NWPP(“NWPP” disclosed as SEQ ID NO: 177) (approximately 6 mg/kg, apreviously-determined sub-efficacious dose), control IgG, 2 mg BIDoseltamivir, or a combination of a single dose of mAb 39.29 NWPP (“NWPP”disclosed as SEQ ID NO: 177) and oseltamivir treatment for 5 days. ALog-rank test of the combination treatment vs. mAb 39.29 NWPP (“NWPP”disclosed as SEQ ID NO: 177) or oseltamivir gives p<0.01.

As expected, control IgG treated animals exhibited 100% mortality 9 dayspost infection. (See FIG. 18.) The mortality observed forcontrol-treated animals was very similar to the groups receiving onlyoseltamivir or a sub-efficacious dose of mAb 39.29 NWPP (“NWPP”disclosed as SEQ ID NO: 177). However, co-administration of asub-efficacious dose of mAb 39.29 NWPP (“NWPP” disclosed as SEQ ID NO:177) plus oseltamivir significantly improved survival compared to thatobserved in either treatment alone, resulting in 87.5% survival. (SeeFIG. 18.)

These results showed that a synergistic effect on the treatment ofinfluenza A virus infection occurred during combination therapy using amonoclonal antibody of the present invention used in combination withoseltamivir, a neuraminidase inhibitor.

Example 10 Anti-Hemagglutinin Antibodies of the Present InventionPerform Better than Oseltamivir in a Ferret H5N1 Influenza a VirusInfection Model

Ferret influenza A virus infection models are often used to examineprophylactic and therapeutic efficacy of anti-influenza therapeutics.Ferrets are considered a clinically relevant animal model for humaninfluenza A virus infection. (See Matsuoka et al., (2009) CurrentProtocols in Microbiology, Chapter 15, Unit 15G 12.)

To examine the in vivo efficacy of mAb 39.29 D8C2 and mAb 81.39 B1C1against a human isolate of H5N1 influenza A virus in ferrets, thefollowing studies were performed. The ferret H5N1 study was completedunder contract at the Lovelace Respiratory Research Institute(Albuquerque, N. Mex.). Male ferrets (Mustela putorius furo) werechallenged with an intranasal dose of 1×10³ pfu of the highly virulentH5N1 A/Vietnam/1203/04 influenza A virus strain (LD90 dose). Animalswere infected 48 or 72 hours prior to receiving antibody by i.v. oroseltamivir (Tamiflu®) by oral gavage. The control treated animalsreceived a 25 mg/kg i.v. dose of mAB gD5237, a monoclonal antibodyspecific for glycoprotein D of HSV. The anti-influenza treated animalsreceived a single 25 mg/kg i.v. dose of either mAb 39.29 D8C2 or mAb81.39 B1C1 at 48 or 72 hours post influenza virus infection. Eachantibody treatment group included 10 ferrets. The oseltamivir treatedanimals received a twice-daily oral dose of 25 mg/kg for 5 days. Animalswere monitored daily for weight loss, fever, and, body conditioning.

Consistent with an H5N1 infection, the majority of infected ferretsshowed early signs of upper respiratory disease by 48 hours postinfection. As expected with a lethal dose of H5N1, the negative controlantibody treatment group exhibited 90% mortality by 14 days postinoculation. (See FIGS. 19A and 19B.)

In contrast, ferrets that received a single dose of mAb 39.29 D8C2 ateither 48 or 72 hours post influenza virus infection showed 80% and 90%survival (20% and 10% mortality), respectively. (See FIG. 19A.)Likewise, ferrets that received a single dose of mAb 81.39 B1C1 ateither 48 or 72 hours post infection showed 100% and 80% survival (0%and 20% mortality), respectively. (See FIG. 19B.) Irrespective oftreatment initiation time, the oseltamivir treated groups showed 50%mortality.

These results showed that broadly neutralizing anti-hemagglutininantibodies of the present invention were highly protective in thetreatment of severe influenza A virus H5N1 infection in ferrets andperformed better than oseltamivir when administered at either 48 and 72hours post influenza A virus infection.

Example 11 Crystallization and Data Collection

In order to examine the structural basis for hemagglutinincross-reactivity of the antibodies of the present invention, mAb 39.29NCv1 Fab fragment was co-cystallized with recombinant hemagglutinin H3from the human influenza A virus strain A/Perth/16/2009 as follows.

Protein Expression and Purification

To better understand the structural basis for hemagglutininneutralization, the crystal structure of mAb 39.29 NCv1 Fab fragment incomplex with hemagglutinin was determined as follows. Nucleic acidencoding the extracellular domain of Perth H3 hemagglutinin (H3HA,A/Perth/16/2009, amino acid residues 25-520 (SEQ ID NO: 226 forfull-length hemagglutinin H3 (H3HA) amino acid sequence) was cloned intopACGP67 vector (BD Biosciences) in-frame with a thrombin cleavage site(LVPRGS, SEQ ID NO: 106), trimerization “foldon” sequence(PGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG, SEQ ID NO:107), and a C-terminal 6×Histag (SEQ ID NO: 108). Recombinant baculovirus was generated byco-transfection of Sf9 cells with the H3HA-pACGP67 vector and linearizedbaculovirus DNA (Pharmingen).

To generate recombinant H3HA protein, Trichoplusia ni PRO cells wereinfected with the recombinant baculovirus using an MOI of 1 and grownfor 72 hours at 27° C. Cell supernatants were treated with 50 mMTris-HCl, pH 7.5, 5 mM CaCl₂, and 1 mM NiCl₂ followed by centrifugationand filtering. Media was then concentrated and buffer exchanged into 10mM Tris, pH 8.0, and 150 mM NaCl (TBS) containing 20 mM imidazole bytangential flow filtration, and protein captured with Ni-agarose andeluted into TBS containing 200 mM imidazole. The foldon tag was cleavedovernight with thrombin, and H3HA was concentrated and further purifiedon a Superdex 200 16/60 size exclusion column equilibrated in TBS.

To generate the hemagglutinin-Fab complex, the mAb 39.29 NCv1 Fab (undercontrol of the PhoA promoter) was expressed in E. coli overnight at 30°C. The cells were pelleted by centrifugation at 6,000 rpm for 15 minutesand lysed by micro-fluidization in PBS supplemented with 25 mM EDTA and1 mM PMSF. Cell debris was removed by centrifugation at 10,000 rpm for 1hour at 4° C. The resulting supernatant was passed through a Protein Gcolumn and Fab eluted with 0.58% acetic acid. Further purification ofmAb 39.29 NCv1 Fab was achieved by SP sepharose chromatography using agradient from 0 to 1 M NaCl in 20 mM MES, pH 5.5. To generate theHA/39.29 complex, H3HA was incubated overnight with excess mAb 39.29NCv1 Fab, followed by concentration and 5200 size exclusionchromatography in TBS to isolate the complex. The complex wasconcentrated to 10 mg/ml for crystallization trials.

Crystallization

Crystal generation for the H3HA/39.29 NCv1 Fab complex were found in0.1M Phosphate/Citrate buffer, pH 4.2, using 40% PEG 300 as precipitant(condition C6, the JCSG+ sparse matrix screen, Qiagen). Diffractionquality crystals were ultimately grown at 19° C. in sitting dropscontaining 0.1 μl protein and 0.1 μl 0.1M Phosphate/Citrate, pH 4.2, 40%PEG 300, and 0.7% 1-butanol. Crystals were cryoprotected in motherliquor followed by flash freezing and storage in liquid nitrogen. Datawas collected under cryo-cooled conditions at the Canadian Light Sourcebeamline CMCF-081D and processed using MOSFLM and SCALA. The crystalbelonged to the I213 space group, with unit cell dimensions ofa=b=c=204.4 and α=β=γ=90°.

Structure Determination

Initial phases were obtained by molecular replacement with PHASER usingthe structure of a H3HA (PDB 3SDY) as a search model. Subsequently theFc and Fv portions of the Fab were placed separately using PHASER, andunderwent initial rounds of rigid body refinement with Phenix. The modelwent through several iterative rounds of adjustment with COOT andsimulated annealing, coordinate, and b-factor refinement with Phenix.Sugar molecules found at Asn-linked glycosylation sites were added usingthe Carboload package from Phenix, and final rounds of refinement werecarried out using REFMAC5. The final model was refined at 3.1 Å withR/Rfree values of 19.9 and 25.9%, respectively. Ramachandran statisticscalculated by Molprobity indicate 89.7% of the residues lie in favoredregions with 1.1% outliers. Contacts were analyzed using the ProteinInterfaces, Surfaces, and Assemblies (PISA) software and structuralfigures were prepared with PYMOL.

Example 12 Structural Characterization of the 39.29 Epitope on H3Hemagglutinin

As described above in Example 11, mAb 39.29 NCv1 Fab fragment wasco-cystallized with recombinant H3 hemagglutinin from the humaninfluenza A virus strain A/Perth/16/2009. The crystal structure of theantibody/hemagglutinin complex was determined at a resolution of 3.1 Å.The overall structure of A/Perth/16/2009 H3 hemagglutinin was similar topreviously determined hemagglutinin structures with the exception ofslight rearrangements and disorder in the HA2 helix 1/helix 2 linker.Disorder at these locations has been seen previously under low pHcrystallization conditions, which is consistent with this complex beingcrystallized at pH 4.2 (Ekiert et al., (2011) Science 333:843-850). Thecrystal structure of the antibody/HA complex showed a single mAb 39.29Fab molecule bound to each monomer of the uncleaved H3 HA trimer. Boththe light chain and heavy chain fragments of mAb 39.29 NCv1 Fabfragments were well resolved throughout, allowing close examination ofthe Fv interaction with HA.

The epitope for mAb 39.29 NCv1 was determined to be on the stalk regionof H3 hemagglutinin, roughly on top of the HA2 helix A. This region ofthe hemagglutinin stalk was first identified as a broadly neutralizingepitope for influenza A viruses expressing Group1 hemagglutinin subtypes(Ekiert et al., (2009) Science 324:246-251; Sui et al., (2009) NatureStructural & Molecular Biology 16:265-273)), and more recently as aneutralizing epitope for influenza A virus strains carrying Group1 andGroup2 hemagglutinin subtypes (Corti et al., (2011) Science333:850-856). mAb 39.29 NCv1 antibody uses extensive heavy and lightchain contacts to bury approximately 1175 Å² of the hemagglutinin stalksurface area. The heavy chain of mAb 39.29 NCv1 contributes to bindinglargely through an extended hydrophobic CDRH3 loop that inserts into ashallow nonpolar groove adjacent to HA2 helix A and underneath aconserved Group2 hemagglutinin glycosylation site at Asn54. This CDRH3loop extends Phe99 side-chain out to interact with H3 hemagglutininThr334, Ile390, and Ile393, while making main chain polar contacts withthe GlcNAc attached to H3 hemagglutinin Asn54. The CDRH3 loop of mAb39.29 NCv1 also makes a β-turn at Gly100, which is likely stabilized byinter-loop main chain contacts between Val98 and Ile100A. Ile100A facesdownward to interact with a conserved H3 hemagglutinin Trp366, whileVal98 and Pro100C also make van der Waals contacts with the H3hemagglutinin stalk. Residing at the heavy/light chain interface,Pro100D and Trp100E terminate the long CDRH3 loop and act to anchor theloop in place.

The light chain of mAb 39.29 NCv1 also contributes significantly to theinteraction with the H3 hemagglutinin stalk, making contacts with the H3hemagglutinin stalk with all three light chain CDR loops as well asframework residues. Of the approximately 1100 Å² hemagglutinin buriedsurface area, ˜60% is contributed by the light chain (640 Å² vs 480 Å²for light chain and heavy chain, respectively). The CDRL1 Asn32 makeshydrogen bond with H3 HA2 helix A residues Asp391 and Asn394, whileCDRL1 His31 stacks against the H3 hemagglutinin Asn376 sidechain. Ser52in the CDRL2 loop also makes a polar contact with Asn398. Within theCDRL3 loop, the backbone of Asn93 contacts Asp391 while Trp94 makes acation-π interaction with Lys384 in the HA2 helix A. Interestingly, mAb39.29 also makes a number of framework contacts with hemagglutinin,primarily through backbone interactions of the SGSGSG repeat (SEQ ID NO:109) in beta-strand 6 of the IgKV3 with amino acid residues 403 to 405in the H3 hemagglutinin polypeptide. Ser67 of mAb 39.29 NCv1 also makespolar interactions with Asp48 and Thr404 of H3 hemagglutinin.

All three mAb 39.89 NCv1 light chain CDR loops contribute to binding ofthe H3 HA stalk epitope, accounting for approximately 60% of the totalburied surface area. This large dependence of light chain contacts isunique among known hemagglutinin Group1 and Group2 binding andneutralizing antibodies, with antibody F16v3 light chain contributing toonly 20% to the buried surface area and antibody CR9114 light chain notmaking contact with the epitope.

Although structurally conserved, Group1 and Group2 hemagglutininsubtypes diverge significantly at the primary amino acid sequence level.To compare mAb 39.29 NCv1 H3HA contact residues with other hemagglutininsubtypes, we aligned the amino acid sequence of H3 hemagglutinin frominfluenza virus A/Perth/16/2009 with representative hemagglutinin aminoacid sequences from other influenza virus strains: H1HA fromA/California/07/2009; H2HA from A/Japan/305/1957; H5HA fromA/Vietnam/1203/2004; and H7HA from A/chicken/NSW/1/1997. The amino acidnumbering of H3 hemagglutinin from A/Perth/16/2009 in the crystalstructure matches the hemagglutinin H3 sequence used in the alignment.The hemagglutinin sequence alignment was generated using clustalW andthe amino acid sequences corresponding to hemagglutinin H1 fromA/California/07/2009, hemagglutinin H2 from A/Japan/305/1957,hemagglutinin H3 from A/Perth/19/2009, hemagglutinin H5 fromA/Vietnam/1203/2004, and hemagglutinin H7 from A/chicken/NSW/1/1997. Thecrystal structure was used to determine the contact residues between the39.29 NCv1 Fab fragment and the stalk of hemagglutinin H3.

The alignment is presented in FIG. 20. Hemagglutinin contact residues(shaded in grey) are defined as residues within 4.5 Å of mAb 39.29 NCv1.Each amino acid residue that had greater than 50% of its availablesurface area buried by mAb 39.29 NCv1 Fab is marked with an asterisk.

A high degree of sequence conservation is observed among the contactresidues that contribute significantly to the binding of mAb 39.29 NCv1to this epitope. (See FIG. 20.) This observation suggests that mAb 39.29NCv1 binds Group1 and Group2 hemagglutinin molecules via the same stalkepitope seen in the crystal structure described above. This epitope issimilar to a hemagglutinin epitope identified for FI6v3anti-hemagglutinin antibody (Corti et al., (2011), supra). However, mAb39.29 NCv1 binds in a different orientation with respect to thehemagglutinin stalk than does FI6v3. Comparison of the 39.29 NCv1,F16v3, and CR9114 structures in complex with HA revealed that all threeantibodies bind an epitope that includes the HA2 helix A and adjacentnon-polar groups. However, each of the three antibodies has a uniquebinding orientation, with each heavy chain bound to a similartopographical position on HA but with light chain positioning rotated by˜60° (F16v3) or ˜120° (CR9114) when compared to 39.29 NCv1. Also uniqueto mAb 39.29 NCv1, the IgKV3 light chain SGSGSG repeat (SEQ ID NO: 109)in beta-strand 6 frame-work makes contact with H3 HA. Therefore, the39.29 structure represents a third solution to the binding of thishighly conserved epitope and solidifies the importance of engaging theHA2 helix A for broad neutralization of influenza A virus.

The crystallography data of mAb 39.29 in complex with H3 hemagglutininfrom the human influenza A virus strain A/Perth/16/2009 revealed thefollowing contact positions: 34, 36, 54, 70, 292, 294, 305, 307, 334,363, 364, 365, 366, 379, 380, 382, 383, 384, 386, 387, 390, 391, 393,394, 395, 397, 398, 401, 403, 404, and 405. Antibody FI6v3 showed thefollowing contact positions: 334, 352, 356, 363, 364, 365, 366, 381,383, 384, 386, 387, 388, 390, 391, 393, 394, 397, 398, 401, and 402.Amino acid residue positions correspond to H3 hemagglutinin frominfluenza A virus strain A/Perth/16/2009 (SEQ ID NO:226). (SeeInternational Application Publication Nos: WO 2010/010466 and WO2013/011347; Corti et al. (2011) Science 333:850-856.) While someoverlap is observed, mAb 39.29 showed a greater number of contactpositions within hemagglutinin than FI6v3.

The fact that mAb 39.29 NCv1 and FI6v3 antibody CDRs have no sequencehomology and that both antibodies engage a similar but not identicalstalk epitope in different ways suggests that there are various ways forantibodies to bind the conserved stalk epitope and broadly neutralizeinfluenza A viruses.

Example 13 Competition ELISA

Competition ELISA assays were developed using hemagglutinin H1 frominfluenza virus A/WSN/1933 and hemagglutinin H3 from influenza virusA/Hong Kong/8/1968. Hemagglutinin-coated ELISA plates were allowed tobind test antibody at various concentrations (X-axis) prior to theaddition of saturating concentrations of biotin labeled mAb 39.29. Ifthe test antibody competed for the hemagglutinin epitope of mAb 39.29,the biotin ELISA signal (Y-axis) was decreased as a function ofincreasing test antibody concentration. The binding data were fit with anon-linear dose response curve to determine the EC₅₀ value given in nM.

mAb 39.29 IgG was biotinylated through amine coupling according to themanufacturer's recommended protocol (Sulfo-NHS-LC-LC, Pierce, Rockford,Ill.). Final stock concentration of the biotinylated mAb was 13.2 mM. Todetermine the optimal concentration for usage, the biotinylated 39.29was serially titrated against immobilized H1 hemagglutinin frominfluenza A virus A/WSN/1933 and H3 hemagglutinin from influenza A virusA/Hong Kong/8/1968. Recombinant hemagglutinin H1 and H3 proteins werediluted to 2 μg/ml in phosphate buffered saline (PBS) and dispensed (100μl) onto 96-well Nunc Maxisorp plates (Nunc, Rochester, N.Y.). Theplates were coated overnight at 4° C., rinsed in PBS, and then blockedfor 1-hour at room temperature with PBS containing 1% bovine serumalbumin (BSA, Sigma-Aldrich, St. Louis, Mo.).

Each plate then received 100 μl of serially diluted biotinylated mAb39.29 starting at an initial concentration of 88 nM with 1/3 dilutionsin PBS containing 1.0% BSA and 0.05% Polysorbate 20 (Sigma-Aldrich).After one hour incubation, the plates were washed and then incubatedwith 100 μl of a 1:5000 dilution of streptavidin-conjugated horseradishperoxidase (Caltag Laboratories, Carlsbad, Calif.) for 30 minutes atroom temperature. Following the incubation, the plates were washed anddeveloped with 100 μl of TMB substrate (Kirkegaard and PerryLaboratories, Inc. Gaithersburg, Md.). Plates were read on a SpectraMaxplate reader (Molecular Devices, Sunnyvale, Calif.) at O.D. 450 nM. Theoptimal concentration of biotinylated mAb was determined to be 1 nM.

Various concentration α-axis) of monoclonal antibodies 39.18, 36.89,81.39 39.29, mAb 9, mAb 23 of the present invention and control IgG wereincubated with the hemagglutinin-coated plates for 30 minutes at roomtemperature. Initial concentration was 200 nM followed by 3 fold serialdilutions. Biotinylated mAb 39.29 was added to a final sub-saturatingconcentration of 1 nM. Following one hour incubation, the plates werewashed and incubated with 100 μl of a 1:5000 dilution ofStreptavidin-conjugated horseradish peroxidase for 45-minutes. Plateswere washed and then develop with TMB solution. If the test antibodycompeted for the HA epitope of mAb 39.29, the biotin ELISA signal(Y-axis) was decreased as a function of increasing test antibodyconcentration. The binding data were fit with a non-linear dose responsecurve to determine the EC₅₀ value given in nM.

FIGS. 21A and 21B show results of competition ELISA analysis of the mAbsfor binding to H1HA from A/NWS/1933 (FIG. 21A) or H3HA from A/HK/8/1968(FIG. 21B). The results showed that mAb 39.29, mAb 81.39, mAb 39.18, andmAb 36.89 all bind to an overlapping hemagglutinin stalk epitope (FIGS.21A and 21B). Specifically, mAb 81.39 and mAb 39.18 compete for bindingof mAb 39.29 on the stalk of hemagglutinin H1 (FIG. 21A), while mAb81.39 and mAb 36.89 compete for binding with mAb 39.29 for theidentified stalk epitope on hemagglutinin H3 (FIG. 21B).

By using competition ELISA assays it was established that monoclonalantibodies 81.39, 39.18, 36.89, mAb 9, and mAb 23 bind to the highlyconserved stalk epitope of hemagglutinin identified by the structuralanalysis. Specifically, the mAb 81.39 and mAb 39.18 compete for bindingof mAb 39.29 on the stalk of the Group1 H1 hemagglutinin. Additionally,mAb 81.39, mAb 36.89, mAb 9, and mAb 23 compete for binding with mAb39.29 for the identified stalk epitope on the Group2 H3 hemagglutinin.As predicted, since mAb 39.18 neutralizes only Group1 Influenza Aisolates, it does not compete for binding of the mAb 39.29 epitope onGroup2 hemagglutinin. Likewise, mAb 36.89, mAb 9, and mAb 23 onlyneutralize Group2 Influenza A isolates and therefore do not compete forbinding of mAb 39.29 on Group1 H1 hemagglutinin. The data from theseexperiments is further summarized in Table 3 below.

TABLE 3 Influenza HA mAb mAb mAb mAb Isolate Subtype 39.18 39.29 81.3936.89 mAb 9 mAb 23 A/NWS/ Grp1/H1 0.88 2.8 2.15 — — — 1933 A/HK/8/Grp2/H3 — 2.54 4.21 1.32 8.42 1.84 1968 EC₅₀ given in nM — IndicatesEC50 > 200 nM

Example 14 Safety and Pharmacokinetics of Anti-Influenza a VirusAntibody in Healthy Volunteers

A phase 1 single-ascending dose study of mAb 39.29-NWPP in healthy humanmale and female subjects 18 years of age or older was performed. Initialdosing to investigate the safety, tolerability, and pharmacokinetics inhealthy adult subjects was performed by i.v. administration of a singledose (1.5 mg/kg, 5 mg/kg, 15 mg/kg, or 45 mg/kg) of mAb39.29. mAb39.29was safe and well-tolerated at all dose levels after a follow-up periodof at least 58 days for the 45 mg/kg dose level and 120 days for the 1.5mg/kg dose level. No serious adverse events related to study drug werereported.

Serum concentrations of mAb 39.29 exhibited a biphasic disposition withan initial rapid distribution phase followed by a slow eliminationphase. mAb39.29 demonstrated linear pharmacokinetics (PK). The meanC_(max) increased in a dose-proportional manner of 33.5 μg/mL for the1.5 mg/kg dose group and 1180 μg/mL for the 45 mg/kg dose group.Similarly, the group mean AUC_(0-infinity) was 518 and 5530 μg/mL*dayfor the 1.5 mg/kg and 15 mg/kg dose groups, respectively, and isapproximately dose proportional. On the basis of the available PK datain healthy male and female subjects, mAb 39.29 appeared to have a PKprofile consistent with that of a typical IgG1 human antibody with amean half-life of approximately 20 days (Mean Range 19.3-22.2).

Example 15 Phase 2 Study of Anti-Influenza a Virus HemagglutininAntibody

A phase 2 clinical study of an anti-influenza A virus hemagglutininantibody of the present invention is performed as follows. Hospitalizedindividuals having influenza A virus infection are administered ananti-influenza A virus hemagglutinin antibody of the present inventionby intravenous administration, at a dose of 1.5 mg/kg, 5 mg/kg, 15mg/kg, or 45 mg/kg. Alternatively, individuals are administered antibodyat a fixed dose of 120 mg, 400 mg, 1200 mg, or 3600 mg. Individuals mayalso be administered oseltamivir (Tamiflu®) (current standard of care)prior to, at the time of, or subsequent to administration of theanti-influenza A virus hemagglutinin antibody. Generally, a one-timedosing regimen of the antibody is used, although subsequent doses arecontemplated.

Administration of an anti-influenza A virus hemagglutinin antibody ofthe present invention shows efficacy at treating influenza A virusinfection, including reduction of influenza A virus infectivity,reduction in the length of hospital stay, reduction or prevention of theneed for intensive care unit use, reduction or prevention of the needfor assisted or mechanical ventilation, or reduction or prevention ofthe need for supplemental oxygen use.

Administration of an anti-influenza A virus hemagglutinin antibody ofthe present invention results shows efficacy at treating influenza Avirus infection by reduction of time to normalization of respiratoryfunction (such as a reduction of time to normalization of respiratoryrate, or a reduction of time to normalization of oxygen saturation),reduction of time to return to normal oxygen saturation, e.g., to anoxygen saturation of about 92% or greater, as measured over a 24 hourperiod without supplemental oxygen administration, or reduction of timeto normalization of vital signs, such as heart rate, blood pressure,respiratory rate, and temperature.

Statistical Analyses

Statistics were calculated using JMP version 9.0.2 software (SASInstitute). Survival experiments were compared using log-rank test. Pvalues<0.05 were considered significant. IC₅₀ curves and values wereplotted and calculated using Graphpad Prism version 5.0 software.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

What is claimed is:
 1. An isolated anti-hemagglutinin monoclonalantibody that specifically binds influenza A virus hemagglutinin,wherein the antibody comprises three heavy chain hypervariable regions(HVR-H1, HVR-H2, and HVR-H3) and three light chain hypervariable regions(HVR-L1, HVR-L2, and HVR-L3), wherein: (a) HVR-H1 comprises the aminoacid sequence of SEQ ID NO:178; (b) HVR-H2 comprises the amino acidsequence of SEQ ID NO:179; (c) HVR-H3 comprises the amino acid sequenceof SEQ ID NO: 181; (d) HVR-L1 comprises the amino acid sequence of SEQID NO: 183; (e) HVR-L2 comprises the amino acid sequence of SEQ IDNO:187; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO:189.
 2. The isolated anti-hemagglutinin antibody of claim 1, wherein theantibody comprises a light chain variable region comprising the aminoacid sequence of SEQ ID NO:
 117. 3. The isolated anti-hemagglutininantibody of claim 1, wherein the antibody comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:
 115. 4.The isolated anti-hemagglutinin antibody of claim 1, wherein theantibody comprises a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises theamino acid sequence of SEQ ID NO: 115, and the light chain variableregion comprises the amino acid sequence of SEQ ID NO:
 117. 5. Theisolated anti-hemagglutinin antibody of claim 1, wherein the antibodycomprises a light chain comprising the amino acid of SEQ ID NO:
 116. 6.The isolated anti-hemagglutinin antibody of claim 1, wherein theantibody comprises a heavy chain comprising the amino acid of SEQ ID NO:114.
 7. The isolated anti-hemagglutinin antibody of claim 1, wherein theantibody comprises a heavy chain and a light chain, wherein the heavychain comprises the amino acid sequence of SEQ ID NO: 114, and the lightchain comprises the amino acid sequence of SEQ ID NO:
 116. 8. Anisolated anti-hemagglutinin monoclonal antibody that specifically bindsinfluenza A virus hemagglutinin, wherein the antibody comprises a heavychain variable region and a light chain variable region, wherein theheavy chain variable region comprises the amino acid sequence of SEQ IDNO:115, and the light chain variable region comprises the amino acidsequence of SEQ ID NO:117.
 9. The isolated anti-hemagglutinin antibodyof claim 8, wherein the antibody comprises a heavy chain and a lightchain, wherein the heavy chain comprises the amino acid sequence of SEQID NO:114, and the light chain comprises the amino acid sequence of SEQID NO:116.
 10. An isolated anti-hemagglutinin monoclonal antibody thatspecifically binds influenza A virus hemagglutinin, wherein the antibodycomprises a heavy chain and a light chain, wherein the heavy chaincomprises the amino acid sequence of SEQ ID NO:114, and the light chaincomprises the amino acid sequence of SEQ ID NO:116.
 11. A compositioncomprising the antibody of claim 1, claim 8, or claim
 10. 12. Apharmaceutical composition comprising the antibody of claim 1, claim 8,or claim 10 and a pharmaceutically acceptable carrier.